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Division of Endocrinology, Metabolism and Diabetes and Program in Human Molecular Biology and Genetics (E.D.A., C.G., S.B., M.P.), University of Utah School of Medicine, Salt Lake City, Utah 84112; Nuffield Department of Clinical Medicine (P.A.R.), University of Oxford, John Radcliffe Hospital II, Headington OX3 9DU, United Kingdom; and Department of Biochemistry (V.K., T.K., Z.X., K.V.K.), Boston University School of Medicine, Boston, Massachusetts 02118
Address all correspondence and requests for reprints to: E. Dale Abel, M.D., Ph.D., Division of Endocrinology, Metabolism and Diabetes, Program in Human Molecular Biology and Genetics, University of Utah School of Medicine, 15 North 2030 East, Building 533, Room 3410B, Salt Lake City, Utah 84112. E-mail: dale.abel{at}hmbg.utah.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Targeted ablation of IRAP leads to a 65–85% reduction in cardiac GLUT4 content (25). Although the mechanism for this observation remains obscure, three possibilities have been proposed (25). 1) Decreased aminopeptidase activity in IRAP-deficient cells may reduce the action of peptide hormones (IRAP substrates) that may play an important role in the regulation of GLUT4 synthesis or degradation. 2) IRAP plays an important role in the sorting and trafficking of GLUT4, so that in its absence GLUT4 is mistargeted and is subsequently degraded. 3) GLUT4 and IRAP may share common degradative pathways and the absence of IRAP leads to accelerated degradation of GLUT4. If the first possibility is correct, then targeted ablation of GLUT4 should not a priori result in reduced IRAP content in muscle and fat cells. However, studies of cardiac and skeletal muscle obtained from GLUT4 null mice revealed 50–60% reductions in IRAP protein content (26). Thus, the likelihood is high that GLUT4 and IRAP are mutually involved in their sorting into and retention within the IRV.
We have addressed these questions by studying mice with selective ablation of GLUT4 in the heart (G4H–/–) (27). By using IRAP and cellugyrin to define the individual intracellular GLUT4-containing vesicular compartments, we have observed that IRAP is partially degraded and/or mistargeted to other membrane fractions. In G4H–/– hearts, IRAP content is reduced by 70% in cellugyrin-positive vesicles (which are likely derived from early endosomes), cellugyrin-negative vesicles (which likely represent the IRV), and on the cell surface (after insulin stimulation). A small IRAP pool translocates to the cell surface, but in sucrose gradients the majority of IRAP was found in the pellet that contains large endosomal and endoplasmic reticulum fragments. Thus, IRAP content is reduced in the IRV, and from early endosome-derived cellugyrin-positive vesicles from which the IRV is believed to arise. These observations suggest that GLUT4 and IRAP are critical for the formation of and/or maintenance of the insulin-responsive vesicle compartment and that absence of one of these protein constituents leads to mistargeting and degradation of the other.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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demonstrates the absence of GLUT4 in total membranes obtained from G4H–/– mice. GLUT4 ablation was associated with approximately 60% reduction in the expression of IRAP. To determine whether the reduction in IRAP occurred at the level of transcription, we examined IRAP mRNA and found no difference in IRAP gene expression in wild-type (WT) and G4H–/– (Fig. 1B). We also measured by Western blotting the expression of several individual proteins that are known to be present in the GLUT4 vesicles (Fig. 1C) and were unable to detect any significant differences in the amount of cellugyrin, IGF-II/mannose-6-phosphate (MPR) or TfRs, secretory carrier-associated membrane proteins (SCAMP) or vesicle-associated membrane protein (VAMP2), in control vs. G4H–/– heart muscle.
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) is enriched in plasma membrane marker proteins but also contains large endosomal fragments and other organelles such as endoplasmic reticulum and cytoskeletal filaments. Figure 2A shows the distribution of GLUT4 and IRAP in sucrose gradient fractions in hearts from fasted WT mice either before or 10 min after the administration of 0.75 U/kg of insulin by ip injection. The pattern of GLUT4 sedimentation is similar to what we have previously observed in skeletal muscle and importantly demonstrates that insulin stimulates a shift in GLUT4 and IRAP from fractions 8–16 (which are enriched in GLUT4 vesicles) to the pellet (P) of the gradient. Thus, intracellular GLUT4-containing vesicles from normal heart muscle behave like GLUT4 vesicles from skeletal muscle and fat (12, 15, 21). These findings are also consistent with data obtained by immunoelectron microscopy (7, 11). In contrast, G4H–/– hearts do not contain GLUT4 (Fig. 2C, G4H–/–). The contaminating band that is seen on top of the specific GLUT4 signal illustrates that both loading of the gradients and the distribution of irrelevant proteins are similar in control and G4H–/– gradients. In G4H–/– hearts, there is marked reduction in the IRAP content of their respective gradient fractions; however, IRAP remains readily detectable in the pellet of the gradient (P).
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, A and B), that was associated with a 30% reduction (P < 0.008) in intracellular IRAP content (Fig. 3C). In contrast, cell surface IRAP expression was reduced by more than 75% in G4H–/– cardiomyocytes. We could detect a 1.5-fold increase (P < 0.003), in cell surface IRAP content in insulin-stimulated G4H–/– cardiomyocytes (Fig. 3, A and B), but in contrast to WT cells we did not detect any significant reduction in intracellular IRAP content in response to insulin stimulation (Fig. 3C). These observations are consistent with translocation of the residual IRAP molecules that remain in the IRV compartment. In WT cells, the ratio of plasma membrane to intracellular IRAP was 1:3 under basal conditions and 2:3 after insulin stimulation. In G4H–/– cells, the ratio of plasma membrane to intracellular IRAP was 1:4 under basal conditions and 1:2 after insulin stimulation. The fact that intracellular IRAP exceeds plasma membrane IRAP in insulin-stimulated cells may have hindered our ability to detect a significant reduction in intracellular IRAP content in insulin-stimulated G4H–/– cells.
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. The distribution of cellugyrin that colocalizes with GLUT4 vesicles as it transits through the early endosomal compartment is similar in G4H–/– and WT hearts. In a similar fashion, the overall distribution MPR and TfR (15, 22, 23) that partially overlaps with that of GLUT4 and IRAP in control heart muscle is not dramatically altered in G4H–/– mice. However, a small reduction in the amount of these receptors in fractions 9–15, where GLUT4-containing vesicles normally sediment, was consistently observed. Cellugyrin-negative GLUT4 vesicles that we believe represent the IRV are 10-fold enriched for IRAP relative to TfR. Thus, the greater depletion of IRAP relative to TfR could be compatible with depletion of IRAP from the IRV compartment in GLUT4-deficient hearts.
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(lane 1), the IRAP content of the supernatant was reduced by approximately 56% (average of three blots) in G4H–/–. The success of the immunoabsorption procedure is evident by the selective yield of cellugyrin-positive vesicles when cellugyrin-coated beads were eluted, and the absence of cellugyrin-positive vesicles in the remaining supernatant. In WT mice there was more GLUT4 in cellugyrin-positive than in cellugyrin-negative vesicles, but the relative distribution of IRAP was similar. In G4H–/–, IRAP content in cellugyrin-positive and -negative vesicles were reduced by approximately 70% relative to WTs. However, the relative distribution of IRAP in cellugyrin-positive and cellugyrin-negative vesicles was similar. These data indicate that IRAP content is reduced both in the IRV and in early endosome-derived cellugyrin-positive vesicles. Given that the bulk of IRAP on sucrose gradients appeared in the pellet in G4H–/–, we are lead to conclude that most of the mistargeted IRAP in G4H–/– cells is sequestered in large endosomal fragments or the endoplasmic reticulum, which are also present in the pellet of the gradient.
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, B and C). These observations suggest that constitutive recycling of the TfR is not altered in GLUT4-deficient cardiomyocytes. As shown in Fig. 6A, there was a trend (P < 0.07) toward reduced cell surface binding of Tf. Given the small reduction of TfRs in GLUT4-enriched fractions of the sucrose gradient (Fig. 4
, G4H–/–), we believe that this modest reduction in Tf binding is compatible with loss of TfRs, from those IRVs that constitutively recycle to plasma membrane (23).
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DISCUSSION |
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If this were the case, then the failure of IRAP to transit from large endosomes to smaller vesicles could indicate a generalized defect in endosomal trafficking. We believe that this is unlikely given the presence of normal Tf recycling indicating that the constitutive endosomal-recycling pathway remains intact. If IRAP is sequestered in the endoplasmic reticulum, the implication is that GLUT4 and IRAP may play important roles in the transit of newly formed proteins to their final destinations in the recycling endosomes and ultimately to the IRV. It is important to emphasize that a small amount of IRAP was present in the IRV compartment of G4H–/–and does translocate to the plasma membrane after insulin stimulation, indicating that the basic machinery of the IRV compartment is present in these cells despite the absence of GLUT4. Our observations are supported by recent data in GLUT4-deficient adipocytes, demonstrating that despite significant reductions in IRAP content, the reduced pool of IRAP could still be translocated to the plasma membranes of these adipocytes in response to insulin stimulation (28). This study also revealed important tissue-specific differences given the fact that in adipocytes, VAMP2 expression appears to be coordinately regulated with that of GLUT4 and IRAP, whereas in contrast we did not observe any relationship between GLUT4 content and VAMP2 in cardiac myocytes.
Unlike the case of constitutively recycling endosomes, IRVs do not immediately travel to the cell surface but are predominantly retained inside the cell (29, 30). The mechanisms that govern the formation and retention of this selective vesicle population are poorly understood and the binding of GLUT4 vesicles to a putative retention molecule has been hypothesized. Our data are consistent with the hypothesis that GLUT4 may directly interact with specific molecules that direct it to the IRV. Precedent for direct interaction between GLUT4 and other intracellular signaling molecules is exemplified by the sentrin-conjugating enzyme mUbc9 that interacts directly with the GLUT4 COOH-terminal intracellular domain and increases GLUT4 content in insulin-responsive GLUT4 vesicles, and more recently by the discovery of TUG that is believed to play a role in the intracellular retention of GLUT4 in insulin-responsive cells under basal conditions (31, 32). Our study therefore provides additional evidence that, in addition to its main biological function as a glucose transporter, GLUT4 may play a key role in its recycling and sorting into the IRV. The coordinate reduction in IRAP content is also consistent with the hypothesis that GLUT4 and IRAP may share common mechanisms for sorting to and retention in the IRV compartment, and the absence of either molecule may lead to mistargeting or increased degradation of the other.
Our results are supported by an earlier study that reported 50 and 60% reduction in IRAP protein content in the skeletal muscle and heart cells, respectively, of mice with global GLUT4 ablation (26). We have extended those observations by showing that the decline in IRAP protein is not occurring at the level of gene transcription, and that the depletion of IRAP is selectively affecting key steps in GLUT4 trafficking. We believe that there is specific degradation of excess IRAP protein, and sequestration of the remaining IRAP protein either in large endosomes or in the endoplasmic reticulum. In contrast with our findings in cardiac muscle, IRAP content in the plasma membranes of skeletal muscle and adipocytes was reported to be increased in mice with global GLUT4 ablation (26). This was based on subcellular fractionation of skeletal muscle into low-density microsomes and plasma membranes (PMs). We have recently showed that this fractionation protocol is associated with significant contamination of these fractions with endosomal membranes (21), which limits the confidence with which the protein content of PMs and endosomes can be reliably distinguished. For this reason, we elected to use a cell-impermeable biotin, which only labels those proteins that are present on the plasma membrane of our cells. Using this approach, we have determined that IRAP is hardly present on the plasma membrane of GLUT4-deficient cardiomyocytes and that constitutive fusion of IRAP with the plasma membrane is not the basis for its mistargeting.
It has been suggested that IRAP can be targeted to an insulin-responsive compartment in cells that do not express GLUT4 (33, 34, 35). Furthermore, there is evidence that the IRV may exist in differentiating 3T3L1 adipocytes before the onset of GLUT4 expression (36). The question therefore arose as to whether or not IRAP completely failed to enter the IRV in GLUT4-deficient cardiomyocytes. Our analysis of cellugyrin-positive and -negative vesicles suggests that IRAP does indeed enter a compartment that likely represents the IRV. If this were not the case, then we would have expected to see markedly reduced or absent IRAP protein in cellugyrin-negative vesicles. Moreover, the significant increase in cell surface biotinylation of IRAP in insulin-stimulated GLUT4-deficient cardiomyocytes also supports the existence of such a pool. These observations are therefore consistent with the model that an insulin-responsive compartment can develop and be maintained in highly insulin-responsive cells such as cardiomyocytes in the absence of GLUT4. Nevertheless, it is clear from our data that in cardiac muscle cells, levels of and intracellular localization of IRAP may be regulated by GLUT4 in vivo. Indeed, recent data from yeast have indicated that functionally related proteins involved in membrane trafficking might mutually regulate their respective protein levels by posttranscriptional mechanisms. Thus, genetic ablation of Sec1p-like/Munc-18, which regulates the interactions between v-SNARE and t (target membrane)-SNARE proteins upon vesicle fusion with the plasma membrane in yeast, results in down-regulation of the t-SNARE protein Tlg2p (37). It is also of relevance that mice with targeted ablation of IRAP exhibit a 50–80% reduction of GLUT4 content in muscle and fat (25). Thus, even in mammalian cells GLUT4 and IRAP fundamentally and mutually regulate their respective protein levels. Therefore, our findings support the view that, although GLUT4 may not be required for the formation of an insulin-responsive compartment, in highly insulin-responsive cells such as muscle and adipose tissue, it is required for the maintenance of IRAP in the GLUT4 recycling compartment(s) and the IRV in vivo. The ultimate proof of this hypothesis will come from reconstitution experiments in which GLUT4 expression is restored to GLUT4-deficient cardiomyocytes. If the hypothesis is correct, it would be expected that IRAP content in recycling early endosomes and the IRV compartment would be restored, when GLUT4 is re-expressed. The absence of these experiments is a limitation of the present study. However, the GLUT4-deficient cardiomyocyte represents an important reagent with which to test this hypothesis in the future.
Regulation of GLUT4 vesicle trafficking is likely to take place on several levels. For example, strong evidence suggests that low-molecular weight GTP-binding proteins of the rab family are also involved in this process (38, 39, 40). In addition, different molecular mechanisms may control exocytosis and internalization of GLUT4 and colocalized proteins (41, 42, 43). Using a differentiated and physiologically relevant cell, our data provide evidence that like the adipocyte, the presence of GLUT4 and/or IRAP are crucial for their sorting into, and maintenance of their cognate vesicular compartment in cardiac muscle cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Antibodies
In the present study, we used the monoclonal anti-GLUT4 antibody 1F8 (44) polyclonal anti-IRAP antibody (15), monoclonal anti-SCAMP antibody 3F8 (45), monoclonal anti-TfR antibody (Zymed Laboratories, San Francisco, CA), diethylaminoethyl-cellulose purified anti-MPR polyclonal antibodies (a kind gift of Dr. M. Czech, University of Massachusetts Medical School, Worcester, MA) and polyclonal anti-VAMP2 antibody (a kind gift of Dr. R. Jahn, Max Plank Institute, Göttingen, Germany). The cellugyrin antibody was raised against the putative cytoplasmic domain of cellugyrin expressed as a glutathione-S-transferase fusion protein.
Membrane Fractionation
The preparation and fractionation of membranes from cardiac muscles was performed as previously described (15). Briefly, hearts were removed, minced, and homogenized on ice for three times (10 sec 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 2000 x g for 10 min and then at 9000 x g for 20 min (P1). The supernatant was then centrifuged at 180,000 x g for 90 min. The 180,000 x g pellet was resuspended in PBS with protease inhibitors, loaded on a 10%–30% (wt/wt) continuous sucrose gradient (3–4 mg of protein per 5-ml gradient) and centrifuged at 48,000 rpm for 55 min in a SW-50.1 rotor. Gradients were fractionated starting from the bottom of the tube. In some experiments, the gradients were divided into 12 fractions and each fraction analyzed by Western blotting (see Fig. 2C). In other experiments, 24 fractions were obtained and every other fraction was analyzed (see Figs. 2A and 4). In both sets of experiments, GLUT4 vesicles were recovered in equivalent fractions of the gradients namely fractions 4–8 (12-fraction gradients) and fractions 8–16 (24-fraction gradients). The pellet of the sucrose gradient centrifugation (P) was resuspended in PBS and analyzed together with the gradient fractions. All centrifugation was performed at 4 C.
Cardiac Myocyte Isolation and Surface Biotinylation
Cardiomyocytes were isolated from the hearts of WT and G4H–/– mice as follows: mice were deeply anesthetized by ip injection of approximately 15 mg of chloral hydrate, and the heart rapidly excised, and arrested in ice-cold buffer (27). The aorta was then cannulated and retrogradely perfused at constant pressure (60 mm Hg) with buffer (in mM) NaCl 126, KCl 4.4, MgCl2 1.0, NaHCO3 4.0, HEPES 10.0, 2,3-butanedione monoxime 30.0, glucose 5.5, pyruvate 1.8, CaCl2 0.04 (pH 7.3), and type I collagenase 0.9 mg/ml, for 8–10 min. The heart was then minced and then sequentially washed in buffer with gradually increasing calcium concentration until a final concentration of 1 mM is achieved. The cells were gently pelleted by centrifugation, resuspended in a modified DMEM (supplemented with 10% fetal calf serum) and plated in laminin-coated tissue culture wells. After waiting for 90 min to allow cardiomyocytes to settle and attach to the laminin-coated plates, the medium was changed to DMEM containing 0.1% BSA for 30 min, after which half of the cells were stimulated with 10 nM insulin for 30 min. Cells were then biotinylated with the cell-impermeable reagent sulfo-N-hydroxysuccinimide-S-S-biotin (Pierce, Rockford, IL) at a final concentration of 0.5 mg/ml for 60 min at 4 C. Cells were then washed three times with ice-cold PBS and subsequently homogenized in HES (HEPES 10 mM, EDTA 5 mM, sucrose 250 mM and 10 µg/ml aprotinin and leupeptin, respectively) + 1% Triton X-100. The homogenate was then incubated overnight with streptavidin beads (Pierce) (46). After a brief centrifugation, the supernatant was saved and beads were washed several times with PBS. The biotinylated proteins were eluted with Laemmli sample buffer containing 10% ß-mercaptoethanol. The eluates (biotinylated proteins) and supernatants (nonbiotinylated proteins) were then loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel and IRAP expression determined by immunoblotting (see Gel Electrophoresis and Immunoblotting).
Immunoabsorption of Cellugyrin-Containing Vesicles
Hearts were homogenized as described above. The heart homogenate was centrifuged at 2000 x g for 10 min and then at 16,000 x g for 20 min. We know from the previous studies that, under these conditions, large endosomal fragments are pelleted and only small vesicles remain in the supernatant (21). Magnetic sheep antirabbit beads (30 µl) were rotated with 2 µg of nonspecific rabbit IgG or affinity purified polyclonal anticellugyrin antibody at 4 C for 4 h in the total volume of 200 µl of PBS. The 16,000 x g supernatant (800 µg each sample) was then added to the beads overnight at 4 C. Unbound material was collected, and the beads were washed three times with PBS. The bound material was eluted with 100 µl 1% Triton in PBS. Eluate (50 µl) along with unbound material (sup, 50 µg) was analyzed by immunoblotting (see Gel Electrophoresis and Immunoblotting).
Measurement of Tf Binding and Uptake
Cell surface binding and internalization of 125I-labeled diferric human Tf (NEN, Boston, MA.) was measured in freshly isolated WT and GLUT4-deficient cardiomyocytes using a modification of previously described protocols (47, 48). Cardiomyocytes were allowed to attach to laminin-coated 12-well plates as described above. The medium was then changed to DMEM (5 mM glucose + 0.1% BSA), and cells were gradually cooled down to 4 C for 10 min. 125ITf (specific activity, 
55 Ci/mmol) was then added to each well to achieve a final concentration of 4 nM, and the cells maintained at 4 C for an additional 60 min. After two washes with ice-cold PBS, cells were lysed by incubating in 1 M NaOH at 37 C for 30 min and the lysate subjected to gamma counting to determine Tf binding. Nonspecific binding was determined in duplicate plates by incubating with excess (2 µM) unlabeled holo Tf (Sigma, St. Louis, MO). After washing with PBS, an independent aliquot of cells was allowed to rewarm to 37 C for 20 min in 500 µl DMEM containing 0.1% BSA. The resulting medium was collected and the Tf activity determined by gamma counting (TfMedium). Cells were then incubated at room temperature in 500 µl of a stripping solution [sodium citrate 20 mM and NaCl 150 mM (pH 5.0)]. The solution was aspirated, and a 400-µl aliquot subjected to gamma counting to determine the amount of 125ITf that remained bound to cardiomyocytes (TfStripped). The sum of TfMedium and TfStripped was used as an indicator of TfR externalization. Finally, after an additional ice-cold PBS wash, cells were lysed by incubation in 600 µl of 1 M NaOH for 20 min, and a 400-µl aliquot of the lysate was subjected to gamma counting to determine Tf uptake and a 40-µl aliquot used for protein determination.
Gel Electrophoresis and Immunoblotting
Protein content was determined with the BCA kit (Pierce) according to manufacturer’s instructions. Proteins were separated in sodium dodecyl sulfate-polyacrylamide gels according to Laemmli (49) and were transferred to Immobilon-P membranes (Millipore, Bedford, MA) in 25 mM Tris, 192 mM glycine. After transfer, the membrane was blocked with 10% nonfat dry milk in PBS for 2 h at 37 C. Proteins were visualized with specific antibodies, horseradish peroxidase-conjugated secondary antibodies (Sigma) and an enhanced chemiluminescence kit (NEN). Autoradiograms were quantitated by laser densitometry (Molecular Dynamics, Sunnyvale, CA).
RNA Analysis
RNA was extracted from cardiac tissue and liver as described (27), and IRAP expression determined using the full-length IRAP cDNA (kind gift of Paul Pilch, Boston University School of Medicine, Boston, MA) as the probe.
Statistical Analysis
Densitometric values for cell surface and intracellular IRAP content, and the results of TfR kinetics were compared by analysis of variance and significance determined at P < 0.05 using the Fisher’s protected least significant difference test.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by research grants from the American Diabetes association and the National Institutes of Health (Grants HL62886 and DK02495) to E.D.A., who is an established investigator of the American Heart Association, by NIH Grants DK52057, DK56736 and by a research grant from the American Diabetes Association (to K.V.K.) and by the Wellcome Trust (to P.A.R.). S.B. was supported by a postdoctoral fellowship from the Association Française pour la Recherche Thérapeutique.
Abbreviations: G4H–/–, Heart-selective GLUT4 null mice; GLUT4, glucose transporter isoform 4; IRAP, insulin-responsive aminopeptidase; IRV, insulin-responsive intracellular membrane vesicle; MPR, IGF-II/mannose 6-phosphate receptor; PM, plasma membrane; Tf, transferrin; TfR, Tf receptor; SCAMP, secretory carrier-associated membrane proteins; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; VAMP2, vesicle-associated membrane protein; WT, wild-type.
Received for publication April 27, 2004. Accepted for publication June 11, 2004.
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, P < 0.001 vs. WT with similar insulin treatment. C, Fold reduction in intracellular (nonbiotinylated) IRAP normalized for total IRAP content (mean ± SE) from nine independent experiments. *, P < 0.0008 vs. non-insulin-treated WT cells; 
