Insulin-mediated translocation of GLUT-4-containing vesicles is preserved in denervated muscles

Min Zhou, Gino Vallega, Konstantin V. Kandror, and Paul F. Pilch

Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Skeletal muscle denervation decreases insulin-sensitive glucose uptake into this tissue as a result of marked GLUT-4 protein downregulation (~20% of controls). The process of insulin-stimulated glucose transport in muscle requires the movement or translocation of intracellular GLUT-4-rich vesicles to the cell surface, and it is accompanied by the translocation of several additional vesicular cargo proteins. Thus examining GLUT-4 translocation in muscles from denervated animals allows us to determine whether the loss of a major cargo protein, GLUT-4, affects the insulin-dependent behavior of the remaining cargo proteins. We find no difference, control vs. denervated, in the insulin-dependent translocation of the insulin-responsive aminopeptidase (IRAP) and the receptors for transferrin and insulin-like growth factor II/mannose 6-phosphate, proteins that completely (IRAP) or partially co-localize with GLUT-4. We conclude that 1) denervation of skeletal muscle does not block the specific branch of insulin signaling pathway that connects receptor proximal events to intracellular GLUT-4-vesicles, and 2) normal levels of GLUT-4 protein are not necessary for the structural organization and insulin-sensitive translocation of its cognate intracellular compartment. Muscle denervation also causes a twofold increase in GLUT-1. In normal muscle, all GLUT-1 is present at the cell surface, but in denervated muscle a significant fraction (25.1 ± 6.1%) of this transporter is found in intracellular vesicles that have the same sedimentation coefficient as GLUT-4-containing vesicles but can be separated from the latter by immunoadsorption. These GLUT-1-containing vesicles respond to insulin and translocate to the cell surface. Thus the formation of insulin-sensitive GLUT-1-containing vesicles in denervated muscle may be a compensatory mechanism for the decreased level of GLUT-4.

denervation; rats; skeletal muscle


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

SKELETAL MUSCLE IS THE MAJOR site for insulin-mediated glucose disposal in humans (7) and rodents (25). The failure of normal insulin levels to stimulate muscle glucose uptake, i.e., insulin resistance, is a major contributor to or cause of type II diabetes mellitus (16, 41). The glucose transporter isoform GLUT-4 is the prime mediator of postprandial muscle glucose uptake (4, 6, 36). GLUT-4 resides in intracellular membrane vesicles in the basal state, and these are translocated to the sarcolemma and T-tubules in response to insulin, thus accounting for the increased clearance of glucose into this tissue (8, 24, 35, 37, 47). In addition to GLUT-4, there is a small amount of GLUT-1 expressed in skeletal muscle, most or all of which is permanently present in the sarcolemma (and/or T-tubules) where it mediates basal glucose transport (12, 47). The molecular basis for insulin-stimulated vesicle movement remains incompletely understood (6) as does the cause for most cases of insulin resistance (16, 41). However, rat hindlimb denervation leads to a type of insulin resistance as a result of marked GLUT-4 downregulation, despite a slight increase in the levels of muscle GLUT-1 (1, 5, 13, 14). Therefore, this model of insulin resistance can be useful in understanding insulin-regulated glucose transport under conditions of altered transporter expression.

In addition to GLUT-4, a number of cargo proteins have been identified as being partially co-localized with GLUT-4 in intracellular vesicles from rat adipocytes, and they translocate to the cell surface in response to insulin. Among these are the insulin-responsive aminopeptidase (IRAP) (20, 23) and sortilin (28, 34), which were identified by microsequencing isolated vesicle proteins after their immunoadsorption with anti-GLUT-4 antibody. The insulin-like growth factor II/mannose 6-phosphate (IGF-II/Man-6-P) and transferrin receptors were identified immunologically (21, 22), as were several other proteins involved in membrane trafficking and fusion, such as secretory component-associated membrane proteins (SCAMPs) (27, 44) and vesicle-associated membrane proteins (VAMPs) (3). We have confirmed that all of the above-named proteins are co-localized with GLUT-4 in vesicles from skeletal muscle (18, 48) and that the sedimentation behavior of vesicles from fat and muscle are identical (18), suggesting that there are no major differences in the composition or trafficking behavior of GLUT-4 in these two tissues.

As noted above, in hindlimb skeletal muscle from the rat, GLUT-4 mRNA and protein expression decrease dramatically after 3 days of denervation. However, there are no notable changes in the levels of other component proteins of GLUT-4-containing vesicles such as IRAP, the transferrin receptor, the IGF-II/Man-6-P receptor, SCAMPs, and VAMP 2 (48). These proteins are still co-localized in the same membrane compartments as in normal extensor digitorum longus (EDL) muscle as determined by immunoadsorption. Thus the denervated muscle represents a type of tissue-specific knockout (or marked reduction) of GLUT-4 and allows us to determine whether this causes an alteration in the behavior of the GLUT-4-deficient vesicles. Here, we show that there is no detectable change in the insulin response of the remaining proteins. Moreover, GLUT-1 shows intracellular sequestration and a modest amount of insulin-responsive translocation to fractions corresponding to the cell surface in apparent compensation for the reduced GLUT-4 levels.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Animals. Male Sprague-Dawley rats (150-175 g) were purchased from Taconic Breeding Laboratory (Germantown, NY). The animals were fasted overnight and then injected with insulin (1.5 units/animal) or with the buffer alone via the portal vein 12 min before being killed, as previously described (5). All rats were anesthetized with pentobarbital sodium (60 mg/kg body wt) by intraperitoneal injection. The EDL muscle was 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. We used monoclonal anti-GLUT-4 antibody 1F8 (17), monoclonal anti-SCAMPs antibody (44), monoclonal anti-transferrin receptor antibody (Zymed Laboratory), monoclonal antidihydropyridine receptor antibody (a kind gift of Dr. Kevin Campbell, University of Iowa), monoclonal anti-VAMP 2 antibody (a kind gift of Dr. R. Jahn, Yale University School of Medicine), O-diethylaminoethyl (DEAE)-cellulose purified anti-IGF-II/Man-6-P receptor polyclonal antibody (a kind gift of Dr. M. Czech, University of Massachusetts Medical School), polyclonal anti-GLUT-1 antibody (a kind gift of Dr. C. Carter-Su, University of Michigan), and polyclonal anti-beta 1 integrin antibodies (a kind gift of Dr. Carles Enrich, Universitat de Barcelona). Polyclonal anti-IRAP antibody was generated by QCB (Hopkinton, MA) in rabbits against synthetic peptide ac-KKSSVPTEEGLIQDEFSC-amide.

Subcellular fractionation of skeletal muscle. Rat EDL muscle (5 muscles/experimental condition) was fractionated as we have recently described (48). Briefly, after cell homogenization and removal of the nonhomogenized tissue by low-speed centrifugation, we carry out a spin at 9,000 g to pellet most of the muscle fibers, cell nuclei, mitochondria, large fragments of sarcoplasmic reticulum, and other heavy subcellular structures and organelles. This pellet (P1) also contains significant amounts of cell surface membrane domains. The membranes in the supernatant from the 9,000-g spin are pelleted by high-speed centrifugation (180,000 g), resuspended in PBS with protease inhibitors [1 µM aprotinin A, 1 µM pepstatin, 1 µM leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF)] loaded on a 10-30% (wt/wt) continuous sucrose gradient (1-1.5 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 formed during gradient centrifugation (P2) contains most of the plasmalemma and T-tubules and is analyzed along with other gradient fractions.

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 concentrations of 0.4 and 0.6 mg of antibodies/ml of resin, respectively, according to the manufacturer's instructions. Before use, 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 and diluted 1:1 with PBS containing 1% BSA. Equal amounts of the total protein from control and denervated muscle samples were incubated with 1F8-coupled beads and nonspecific IgG-coupled beads overnight at 4°C. The beads were washed 4 times with PBS and once with 10 mM Tris, pH 7.4, and the adsorbed material was then eluted with SDS-PAGE sample buffer (26) without 2-mercaptoethanol to avoid dissociation of immobilized IgG.

Muscle biotinylation. Male Sprague-Dawley rats (50-100 g) were fasted overnight, and EDL muscles (4/experimental condition) 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. (31). 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 cleavable EZ-Link sulfo-NHS-SS-biotin (Pierce) was added to a final concentration of 0.5 mg/ml. Biotinylation was performed for 20 min at 37°C, and the reaction was stopped by adding 1 M Tris, pH 7.4, to a final concentration 50 mM. After 3 washes with 50 mM Tris, pH 7.4, muscles were then minced and homogenized on ice three times (10 s each) with 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 PMSF, pH 7.4. 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 the total protein) was incubated with 30 µl of 50% slurry of ImmunoPure Immobilized Streptavidin overnight at 4°C with rotation. The beads were washed three times with 1% Triton X-100 in PBS, and biotinylated proteins were eluted with Laemmli sample buffer containing 2-mercaptoethanol.

Gel electrophoresis and immunoblotting. Protein samples were electrophoresed according to Laemmli (26) and transferred to an Immun-Blot polyvinylidene fluoride membrane (Bio-Rad). 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 conjugated to horseradish peroxidase. Blots were developed by means of an enhanced chemiluminescence detection system (Du Pont NEN), and films were scanned with a computing densitometer (Molecular Dynamics) for quantitative analysis.

Protein content. Protein concentration was determined with the BCA kit (Pierce) according to manufacture's instructions.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

GLUT-4-vesicles translocate to the cell surface in response to insulin in control and denervated EDL muscle as detected by subcellular fractionation. Three days after denervation of the sciatic nerve, rats were injected with insulin or buffer via the portal vein. Control and denervated EDL muscles from the same rat were removed 12 min after injection and fractionated, as described in EXPERIMENTAL PROCEDURES. As we have previously demonstrated, the intracellular pools of recycling proteins are present in the gradient fractions, whereas pellets P1 and P2 contain the cell surface domains (plasma membrane and T-tubules) where GLUT-4 and other proteins are translocated on insulin stimulation. As shown in Fig. 1, GLUT-4 protein expression in denervated EDL muscle is decreased in intracellular membrane vesicles (gradient fractions) and in all surface membrane (P1 and P2) compared with controls, which is consistent with the published data from our own and other laboratories cited in the introductory section. The sedimentation coefficient of GLUT-4-containing vesicles is not changed after denervation. Interestingly, the effect of denervation on movement of GLUT-4 to the P2 fraction appears greater than that for the P1 fraction for unknown reasons.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 1.   GLUT-4 translocates to cell surface in response to insulin in control and denervated extensor digitorum longus (EDL) muscle. Control and denervated EDL muscles from same rat were taken 12 min after insulin treatment and homogenized, and muscle membranes were fractionated (see EXPERIMENTAL PROCEDURES). A: proportional amounts (80 µg) of pellets P1 and P2, and gradient fractions (30 µl each) were analyzed by Western blotting with horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence followed by film exposure. Representative result of 5 independent experiments is shown. B: data from A were scanned in densitometer and subjected to quantitative analysis. Squares, basal muscle; circles, insulin-stimulated; open, control; filled, denervated. A.U., arbitrary units. Results for fractions corresponding to cell surface membranes are expressed as relative percentage; data from P1 and P2 fractions from control muscle (no insulin, no denervation) are each assigned value of 100. Results are means ± SE of 3 separate experiments. * P < 0.05, significant difference from basal values according to Student's t-test.

Insulin administration causes a profound decrease in GLUT-4 content in the fractions of the gradient and a corresponding enrichment in P1 and P2 in both control and denervated EDL muscle (Fig. 1). A similar effect of insulin can also be seen in the case of other major component proteins of GLUT-4-vesicles, IRAP, the transferrin receptor, and the IGF-II/Man-6-P, receptor (Fig. 2). As we have previously shown, almost all IRAP and a considerable amount of the two receptors co-localize with GLUT-4 in normal muscle, and they move to the surface membranes in response to insulin (48). Thus the data of Fig. 2 suggest that, in denervated EDL muscle, insulin can induce translocation of the GLUT-4-containing vesicular compartment to the cell surface despite the reduced amount of GLUT-4.




View larger version (293233K):
[in this window]
[in a new window]
 
Fig. 2.   Component proteins of GLUT-4-containing vesicles: (A) insulin-responsive aminopeptidase (IRAP), (B) transferrin (TfR), and (C) insulin-like growth factor II/mannose 6-phosphate (IGF-II/Man-6-P; IGFIIR) receptors translocate to cell surface in response to insulin in control and denervated EDL muscle. Control and denervated EDL muscles membranes were obtained and fractionated (see EXPERIMENTAL PROCEDURES and Fig. 1). A-C: Western blot representative of three independent experiments. Symbols and quantitative analysis are as in Fig. 1. P1 and P2 fractions were set to 100%. Results are means ± SE of 3 separate experiments. * P < 0.05, significant difference from basal values according to Student's t-test.

GLUT-4-vesicles translocate to the cell surface in response to insulin in control and denervated EDL muscle as detected by cell surface biotinylation. Cell impermeable biotinylation reagents, such as sulfo-NHS-biotin and cleavable sulfo-NHS-SS-biotin, have been used for the analysis of protein traffic in insulin-sensitive tissues in our and other laboratories (19, 39). These reagents react with lysine residues and/or amino termini in the extracellular domains of cell surface proteins, and they do not react with intracellular proteins unless the latter move to the cell surface. Thus increased biotinylation of an intracellular membrane protein, stimulated by insulin, for example, strongly indicates its translocation to the site accessible for biotinylation, i.e., the cell surface. Thus control and denervated EDL muscles were incubated with sulfo-NHS-SS-biotin in the absence and in the presence of insulin and were then processed for electrophoresis, as described in EXPERIMENTAL PROCEDURES. As shown in Fig. 3A, insulin treatment of EDL muscle increases the specific biotinylation of the transferrin receptor by 48 ± 11% in control EDL and 51 ± 8.3% in denervated EDL. Insulin also stimulated biotinylation of IRAP by 26 ± 4.2% in control EDL and 33 ± 7.1% in denervated EDL. These data confirm the results of Fig. 2 and document that insulin's effect on translocation of IRAP and the transferrin receptor is not changed in denervated EDL compared with normal muscle. We could not use this approach in the case of GLUT-4 and the IGF-II/Man-6-P receptor, because GLUT-4 is biotinylated to a very low degree, most probably because there is only one lysine in its extracellular portion that is minimally accessible for reaction, whereas antigenicity of the IGF-II/Man-6-P receptor was destroyed by 2-mercaptoethanol.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   IRAP and transferrin receptors translocate to cell surface in response to insulin in control and denervated EDL muscle, as revealed by in vitro cell surface biotinylation. Isolated EDL muscles from control and denervated animals were incubated with sulfo-NHS-SS-biotin in absence and in presence of insulin and were processed as in EXPERIMENTAL PROCEDURES. An aliquot of Triton X-100 supernatant from microcentrifuge spin (1 mg) was incubated with ImmunoPure Immobilized Streptavidin overnight at 4°C with rotating. Biotinylated proteins adsorbed by Streptavidin beads were eluted from them with Laemmli sample buffer containing 2-mercaptoethanol (2, 29). Eluates were analyzed by Western blot with antibodies to IRAP and transferrin receptor. A: Western blot analysis; B: quantitation of results, expressed as relative percentage with data from muscle without insulin treatment having assigned value of 100. Results are means ± SE of 3 separate experiments. * P < 0.05, significant difference from basal values according to Student's t-test.

Formation of intracellular GLUT-1-containing vesicles in denervated EDL muscle. As noted in the introductory section, it has previously been shown that the expression of GLUT-1, unlike that of GLUT-4, is increased in denervated muscles. However, the subcellular distribution of the de novo synthesized GLUT-1 protein has not been addressed in these previous studies. Figure 4A demonstrates that, in control EDL muscle, GLUT-1 is present almost exclusively at the cell surface. In denervated EDL, the amount of GLUT-1 is greatly increased, and a significant fraction of these transporters (25.1 ± 6.1%) is subsequently found in the part of the gradient corresponding to intracellular vesicles. These data suggest that GLUT-1 may be targeted to a specific intracellular vesicular compartment in denervated muscles that does not exist in normal muscle. As is the case for fat cells (18), GLUT-1-containing vesicles have a sedimentation coefficient indistinguishable from GLUT-4-containing vesicles (Fig. 4B). However, when we performed immunoadsorption with anti-GLUT-4 monoclonal antibody 1F8, we found that GLUT-1 is largely excluded from GLUT-4-containing vesicles. As shown in Fig. 5, under conditions when 88 ± 4.1% of GLUT-4 is immunoadsorbed, no GLUT-1 is found in the eluate from immunobeads. We thus conclude that in denervated EDL muscle, GLUT-1 is not targeted to GLUT-4-containing vesicles, but rather to a different vesicular population.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 4.   Expression of GLUT-1 is upregulated in intracellular vesicles and at cell surface in denervated EDL muscle. Muscle membranes from control and denervated EDL from same rat were prepared and analyzed for GLUT-1 by Western blotting (see EXPERIMENTAL PROCEDURES). A: representative result of 3 independent experiments; B: quantitation of results with horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence. Squares, GLUT-4; circles, GLUT-1; filled, control EDL; open, denervated muscle. GLUT-4 distribution in gradients from control EDL is shown as reference. Results are expressed as relative percentage with data from control muscle having assigned value of 100. Results are means ± SE of 3 separate experiments. * P < 0.05, significant difference from basal values according to Student's t-test.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5.   GLUT-1 is excluded from GLUT-4-containing vesicles in denervated EDL muscle. Membrane protein (500 µg) from denervated muscle from supernatant of 9,000-g centrifugation step was incubated with 100 µl 1F8-coupled beads or nonspecific IgG-coupled beads overnight at 4°C. Immunobeads were washed and then eluted with Laemmli sample buffer without 2-mercaptoethanol and analyzed by Western blotting. Equal amounts of original material from normal and denervated EDL are loaded. Note that original material of normal and denervated EDL used for Western blot is half that used for immunoadsorption. A: Western blot analysis with antibodies to GLUT-4 and GLUT-1; representative result of 3 independent experiments is shown. B: quantitation of results, which are expressed as relative percentage with data from original material having assigned value of 100. Results are means ± SE of 3 separate experiments. ** P < 0.01, significant difference from basal values according to Student's t-test.

Insulin causes translocation of GLUT-1-containing vesicles in denervated EDL muscle. We then asked whether or not these GLUT-1-containing vesicles respond to insulin in denervated EDL. Using the same centrifugation protocol for monitoring the translocation of GLUT-4-containing vesicles, we examined the translocation of GLUT-1-containing vesicles in the denervated EDL muscle. The results are presented in Fig. 6, where insulin stimulation is shown to cause a decrease of intracellular GLUT-1-containing vesicles and an increased level of GLUT-1 protein in the fractions corresponding to the cell surface. It is clear that these GLUT-1-containing vesicles respond to the signals generated by insulin. However, the extent of translocation of these vesicles is much less than that of GLUT-4-containing vesicles, and the nature and composition of the GLUT-1-containing vesicles remain to be determined.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 6.   GLUT-1-containing vesicles are insulin responsive in denervated EDL muscle. Denervated EDL muscles were removed from animals 12 min after insulin treatment and then homogenized. Muscle membranes were fractionated (see EXPERIMENTAL PROCEDURES). A: representative result of 3 independent experiments; B: quantitation of results with horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have previously demonstrated that denervation of hindlimb muscle causes a considerable downregulation of GLUT-4 protein, whereas the levels of expression of other component proteins of GLUT-4-vesicles remain the same (48). As is noted in the introductory section, many investigators have shown that GLUT-4 expression is downregulated by denervation and that insulin-sensitive glucose uptake is markedly attenuated (1, 5, 13, 14). The question remained, however, as to whether the lack of an insulin response in denervated muscle was due mainly to GLUT-4 downregulation (32) or whether there were signaling (see below) and/or vesicular trafficking defects as well. Thus we show that, in denervated EDL muscle, GLUT-4 vesicles maintain insulin responsiveness and are translocated to the cell surface after insulin administration. As previously suggested (13), the marked decrease in the GLUT-4 protein in denervated muscle may be partially compensated by increased expression of GLUT-1, which is expressed both at the cell surface and in intracellular vesicles that have a sedimentation coefficient indistinguishable from GLUT-4-containing vesicles but can be separated from the latter by immunoadsorption.

Thus we believe that the normal level of GLUT-4 is not necessary for insulin-sensitive translocation of its cognate intracellular compartment in skeletal muscle. This conclusion is consistent with our previous studies that show that, during adipocyte differentiation, the formation of insulin-responsive vesicles precedes expression of GLUT-4 (10). These data also suggest that the presence of GLUT-4 in vesicles may not be absolutely necessary for the formation and functioning of this compartment. Additional evidence for this conclusion comes from studies using transgenic animals, where GLUT-4-vesicles from fat were shown to accommodate much more GLUT-4 protein than in normal tissue without any major changes in their composition and insulin responsiveness (45). Thus the specific content of GLUT-4 protein in vesicles may vary to a considerable degree in both directions without major losses in physiological functions of this compartment.

Our results also indicate that the insulin signaling pathway that leads to translocation of GLUT-4 vesicles is not markedly affected by denervation, and the loss of GLUT-4 is, therefore, likely to be the major reason for reduced insulin-stimulated glucose uptake under this physiological condition. It has been shown, however, that insulin-dependent regulation of glycogen synthesis and amino acid transport is significantly impaired early after denervation (42, 43, 46). Those studies showed that the effect of denervation on these processes in skeletal muscle is dependent on muscle fiber type and time after denervation. Thus the reduced insulin-mediated glucose uptake due to the loss of GLUT-4 expression may contribute to the decreased insulin-stimulated glycogen synthesis seen in denervated muscle, but there are likely to be other defects in insulin action that are caused by denervation. Presently, it is not clear where insulin-dependent signal transduction might diverge to glycogen synthesis on the one hand and GLUT-4 translocation on the other. It is clear, however, that GLUT-4 translocation in skeletal muscle can be regulated by exercise independently of the insulin signaling pathway (11).

Basal glucose uptake in denervated muscles is increased, which is consistent with our data showing upregulation of GLUT-1 at the cell surface (Fig. 4). A significant fraction of this transporter (25.1 ± 6.1%) is, however, found in intracellular vesicles. In rat adipose tissues, unlike skeletal muscle, about half of GLUT-1 resides at the plasma membrane in the basal state with the rest of GLUT-1 being sequestered inside the cells where it can translocate to the cell surface in response to insulin (15, 49). The component proteins of these vesicles besides GLUT-1 are unknown; it is also unknown whether the GLUT-1-containing vesicles in denervated skeletal muscle are the same as those in adipocytes. However, because the GLUT-1-containing vesicles in denervated EDL are insulin responsive, they may be useful for studying signal transduction to a vesicle population in the absence of a large background of GLUT-4-containing vesicles.

As is noted several places elsewhere in this paper, GLUT-4 and GLUT-1 protein and mRNA levels are differentially affected by denervation. The transcriptional mechanisms that underlie this phenomenon remain unknown but may involve myocyte-specific enhancer factors in the case of GLUT-4 (30). During muscle development in vivo (40), as well as a function of the differentiation state of cultured muscle cell lines (33, 38), the expression of GLUT-1 decreases, and GLUT-4 concomitantly increases. In denervated muscle, the regulation of these two transporters is reversed; moreover, denervation is associated with the upregulation of several myogenic transcription factors as well as the alpha -subunit of acetylcholine receptor, and these same proteins are downregulated during postnatal development (9). Thus denervation may induce partial dedifferentiation of muscle due to the absence of selective gene regulation by nerve stimuli and may represent an interesting experimental model for studying the developmental acquisition of specific insulin responses.


    ACKNOWLEDGEMENTS

We are grateful to Dr. D. J. Dean and T. G. Kurowski for methodological advice in muscle denervation and in vitro incubation. We also thank Dr. Amr El Jack for help and discussion during the experiments and Dr. T. A. Kupriyanova for providing antibody-coupled beads for immunoadsorption experiments.


    FOOTNOTES

This work was supported by research grants from National Institutes of Health (DK-30425 to P. F. Pilch, DK-49147 to N. Ruderman, and DK-52057 to K. V. Kandror) and by Grant 197029 from the Juvenile Diabetes Foundation and 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 and other correspondence: P. F. Pilch, Department of Biochemistry, Boston University School of Medicine, 715 Albany St., Boston, MA 02118 (E-mail: pilch{at}biochem.bumc.bu.edu).

Received 25 October 1999; accepted in final form 22 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Block, NE, Menick DR, Robinson KA, and Buse MG. Effect of denervation on the expression of two glucose transporter isoforms in rat hindlimb muscle. J Clin Invest 88: 1546-1552, 1991[ISI][Medline].

2.   Buckie, JW, and Cook GM. Specific isolation of surface glycoproteins from intact cells by biotinylated concanavalin A and immobilized streptavidin. Anal Biochem 156: 463-472, 1986[ISI][Medline].

3.   Cain, CC, Trimble WS, and Lienhard GE. Members of the VAMP family of synaptic vesicle proteins are components of glucose transporter-containing vesicles from rat adipocytes. J Biol Chem 267: 11681-11684, 1992[Abstract/Free Full Text].

4.   Charron, MJ, Katz EB, and Olson AL. GLUT4 gene regulation and manipulation. J Biol Chem 274: 3253-3256, 1999[Free Full Text].

5.   Coderre, L, Monfar MM, Chen KS, Heydrick SJ, Kurowski TG, Ruderman NB, and Pilch PF. Alteration in the expression of GLUT-1 and GLUT-4 protein and messenger RNA levels in denervated rat muscles. Endocrinology 131: 1821-1825, 1992[Abstract].

6.   Czech, MP, and Corvera S. Signaling mechanisms that regulate glucose transport. J Biol Chem 274: 1865-1868, 1999[Free Full Text].

7.   DeFronzo, RA, Jacot E, Jequier E, Maeder E, Wahren J, and Felber JP. The effect of insulin on the disposal of intravenous glucose. Results from indirect calorimetry and hepatic and femoral venous catheterization. Diabetes 30: 1000-1007, 1981[ISI][Medline].

8.   Dohm, GL, Dolan PL, Frisell WR, and Dudek RW. Role of transverse tubules in insulin stimulated muscle glucose transport. J Cell Biochem 52: 1-7, 1993[ISI][Medline].

9.   Duclert, A, Piette J, and Changeux JP. Influence of innervation of myogenic factors and acetylcholine receptor alpha-subunit mRNAs. Neuroreport 2: 25-28, 1991[ISI][Medline].

10.   El-Jack, AK, Kandror KV, and Pilch PF. The formation of an insulin-responsive vesicular cargo compartment is an early event in 3T3-L1 adipocyte differentiation. Mol Biol Cell 10: 1581-1594, 1999[Abstract/Free Full Text].

11.   Goodyear, LJ, and Kahn BB. Exercise, glucose transport, and insulin sensitivity. Annu Rev Med 49: 235-261, 1998[ISI][Medline].

12.   Gulve, EA, Ren JM, Marshall BA, Gao J, Hansen PA, Holloszy JO, and Mueckler M. Glucose transport activity in skeletal muscles from transgenic mice overexpressing GLUT1. Increased basal transport is associated with a defective response to diverse stimuli that activate GLUT4. J Biol Chem 269: 18366-70, 1994[Abstract/Free Full Text].

13.   Handberg, A, Megeney LA, McCullagh KJ, Kayser L, Han X-X, and Bonen A. Reciprocal GLUT-1 and GLUT-4 expression and glucose transport in denervated muscles. Am J Physiol Endocrinol Metab 271: E50-E57, 1996[Abstract/Free Full Text].

14.   Henriksen, EJ, Rodnick KJ, Mondon CE, James DE, and Holloszy JO. Effect of denervation or unweighting on GLUT-4 protein in rat soleus muscle. J Appl Physiol 70: 2322-2327, 1991[Abstract/Free Full Text].

15.   Holman, GD, Kozka IJ, Clark AE, Flower CJ, Saltis J, Habberfield AD, Simpson IA, and Cushman SW. Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester. J Biol Chem 265: 18172-18179, 1990[Abstract/Free Full Text].

16.   Hunter, SJ, and Garvey WT. Insulin action and insulin resistance: diseases involving defects in insulin receptors, signal transduction, and the glucose transport effector system. Am J Med 105: 331-345, 1998[ISI][Medline].

17.   James, DE, Brown R, Navarro J, and Pilch PF. Insulin-regulatable tissues express a unique insulin-sensitive glucose transport protein. Nature 333: 183-185, 1988[ISI][Medline].

18.   Kandror, KV, Coderre L, Pushkin AV, and Pilch PF. Comparison of glucose-transporter-containing vesicles from rat fat and muscle tissues: evidence for a unique endosomal compartment. Biochem J 307: 383-390, 1995[ISI][Medline].

19.   Kandror, KV, and Pilch PF. Identification and isolation of glycoproteins that translocate to the cell surface from GLUT4-enriched vesicles in an insulin-dependent fashion. J Biol Chem 269: 138-142, 1994[Abstract/Free Full Text].

20.   Kandror, KV, Yu L, and Pilch PF. The major protein of GLUT4-containing vesicles, gp160, has aminopeptidase activity. J Biol Chem 269: 30777-30780, 1994[Abstract/Free Full Text].

21.   Kandror, KV, and Pilch PF. The insulin-like growth factor II/mannose 6-phosphate receptor utilizes the same membrane compartments as GLUT4 for insulin-dependent trafficking to and from the rat adipocyte cell surface. J Biol Chem 271: 21703-21708, 1996[Abstract/Free Full Text].

22.   Kandror, KV, and Pilch PF. Multiple endosomal recycling pathways in rat adipose cells. Biochem J 331: 829-835, 1998[ISI][Medline].

23.   Keller, SR, Scott HM, Mastick CC, Aebersold R, and Lienhard GE. Cloning and characterization of a novel insulin-regulated membrane aminopeptidase from Glut4 vesicles. J Biol Chem 270: 23612-23618, 1995[Abstract/Free Full Text].

24.   Klip, A, Ramlal T, Young DA, and Holloszy JO. Insulin-induced translocation of glucose transporters in rat hindlimb muscles. FEBS Lett 224: 224-230, 1987[ISI][Medline].

25.   Kraegen, EW, James DE, Jenkins AB, and Chisholm DJ. Dose-response curves for in vivo insulin sensitivity in individual tissues in rats. Am J Physiol Endocrinol Metab 248: E353-E362, 1985[Abstract/Free Full Text].

26.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[ISI][Medline].

27.   Laurie, SM, Cain CC, Lienhard GE, and Castle JD. The glucose transporter Glut4 and secretory carrier membrane proteins (SCAMPs) colocalize in rat adipocytes and partially segregate during insulin stimulation. J Biol Chem 268: 19110-19117, 1993[Abstract/Free Full Text].

28.   Lin, BZ, Pilch PF, and Kandror KV. Sortilin is a major protein component of Glut4-containing vesicles. J Biol Chem 272: 24145-24147, 1997[Abstract/Free Full Text].

29.   Lisanti, MP, Le Bivic A, Sargiacomo M, and Rodriguez-Boulan E. Steady-state distribution and biogenesis of endogenous Madin-Darby canine kidney glycoproteins: evidence for intracellular sorting and polarized cell surface delivery. J Cell Biol 109: 2117-2127, 1989[Abstract].

30.   Liu, ML, Olson AL, Edgington NP, Moye-Rowley WS, and Pessin JE. Myocyte enhancer factor 2 (MEF2) binding site is essential for C2C12 myotube-specific expression of the rat GLUT4/muscle-adipose facilitative glucose transporter gene. J Biol Chem 269: 28514-28521, 1994[Abstract/Free Full Text].

31.   Maizels, EZ, Ruderman NB, Goodman MN, and Lau D. Effect of acetoacetate on glucose metabolism in the soleus and extensor digitorum longus muscles of the rat. Biochem J 162: 557-568, 1977[ISI][Medline].

32.   Megeney, LA, Neufer PD, Dohm GL, Tan MH, Blewett CA, Elder GC, and Bonen A. Effects of muscle activity and fiber composition on glucose transport and GLUT-4. Am J Physiol Endocrinol Metab 264: E583-E593, 1993[Abstract/Free Full Text].

33.   Mitsumoto, Y, Burdett E, Grant A, and Klip A. Differential expression of the GLUT1 and GLUT4 glucose transporters during differentiation of L6 muscle cells. Biochem Biophys Res Commun 175: 652-659, 1991[ISI][Medline].

34.   Morris, NJ, Ross SA, Lane WS, Moestrup SK, Petersen CM, Keller SR, and Lienhard GE. Sortilin is the major 110-kDa protein in GLUT4 vesicles from adipocytes. J Biol Chem 273: 3582-3587, 1998[Abstract/Free Full Text].

35.   Munoz, P, Rosenblatt M, Testar X, Palacin M, Thoidis G, Pilch PF, and Zorzano A. The T-tubule is a cell-surface target for insulin-regulated recycling of membrane proteins in skeletal muscle. Biochem J 312: 393-400, 1995[ISI][Medline].

36.   Pessin, JE, Thurmond DC, Elmendorf JS, Coker KJ, and Okada S. Molecular basis of insulin-stimulated GLUT4 vesicle trafficking. Location! Location! Location! J Biol Chem 274: 2593-2596, 1999[Free Full Text].

37.   Ploug, T, van Deurs B, Ai H, Cushman SW, and Ralston E. Analysis of GLUT4 distribution in whole skeletal muscle fibers: identification of distinct storage compartments that are recruited by insulin and muscle contractions. J Cell Biol 142: 1429-1446, 1998[Abstract/Free Full Text].

38.   Richardson, JM, and Pessin JE. Identification of a skeletal muscle-specific regulatory domain in the rat GLUT4/muscle-fat gene. J Biol Chem 268: 21021-21027, 1993[Abstract/Free Full Text].

39.   Ross, SA, Keller SR, and Lienhard GE. Increased intracellular sequestration of the insulin-regulated aminopeptidase upon differentiation of 3T3-L1 cells. Biochem J 330: 1003-1008, 1998[ISI][Medline].

40.   Santalucia, T, Camps M, Castello A, Munoz P, Nuel A, Testar X, Palacin M, and Zorzano A. Developmental regulation of GLUT-1 (erythroid/Hep G2) and GLUT-4 (muscle/fat) glucose transporter expression in rat heart, skeletal muscle, and brown adipose tissue. Endocrinology 130: 837-846, 1992[Abstract].

41.   Shepherd, PR, and Kahn BB. Glucose transporters and insulin action---implications for insulin resistance and diabetes mellitus. N Engl J Med 341: 248-257, 1999[Free Full Text].

42.   Smith, RL, and Lawrence JC, Jr. Insulin action in denervated rat hemidiaphragms. Decreased hormonal stimulation of glycogen synthesis involves both glycogen synthase and glucose transport. J Biol Chem 259: 2201-2207, 1984[Abstract/Free Full Text].

43.   Sowell, MO, Dutton SL, and Buse MG. Selective in vitro reversal of the insulin resistance of glucose transport in denervated rat skeletal muscle. Am J Physiol Endocrinol Metab 257: E418-E425, 1989[Abstract/Free Full Text].

44.   Thoidis, G, Kotliar N, and Pilch PF. Immunological analysis of GLUT4-enriched vesicles. Identification of novel proteins regulated by insulin and diabetes. J Biol Chem 268: 11691-11696, 1993[Abstract/Free Full Text].

45.   Tozzo, E, Kahn BB, Pilch PF, and Kandror KV. Glut4 is targeted to specific vesicles in adipocytes of transgenic mice overexpressing Glut4 selectively in adipose tissue. J Biol Chem 271: 10490-10494, 1996[Abstract/Free Full Text].

46.   Turinsky, J. Dynamics of insulin resistance in denervated slow and fast muscles in vivo. Am J Physiol Regulatory Integrative Comp Physiol 252: R531-R537, 1987[Abstract/Free Full Text].

47.   Wang, W, Hansen PA, Marshall BA, Holloszy JO, and Mueckler M. Insulin unmasks a COOH-terminal Glut4 epitope and increases glucose transport across T-tubules in skeletal muscle. J Cell Biol 135: 415-430, 1996[Abstract].

48.   Zhou, M, Sevilla L, Vallega G, Chen P, Palacin M, Zorzano A, Pilch PF, and Kandror KV. Insulin-dependent protein trafficking in skeletal muscle cells. Am J Physiol Endocrinol Metab 275: E187-E196, 1998[Abstract/Free Full Text].

49.   Zorzano, A, Wilkinson W, Kotliar N, Thoidis G, Wadzinkski BE, Ruoho AE, and Pilch PF. Insulin-regulated glucose uptake in rat adipocytes is mediated by two transporter isoforms present in at least two vesicle populations. J Biol Chem 264: 12358-12363, 1989[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 278(6):E1019-E1026
0193-1849/00 $5.00 Copyright © 2000 the American Physiological Society