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Originally published In Press as doi:10.1074/jbc.M710604200 on February 7, 2008

J. Biol. Chem., Vol. 283, Issue 15, 10208-10220, April 11, 2008
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Functional Role of Sortilin in Myogenesis and Development of Insulin-responsive Glucose Transport System in C2C12 Myocytes*

Miyako Ariga{ddagger}§1, Taku Nedachi§, Hideki Katagiri{ddagger}, and Makoto Kanzaki§2

From the {ddagger}21st Century COE program Comprehensive Research and Education Center for Planning of Drug Development and Clinical Evaluation, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8575, the §Division of Biomaterials, Biomedical Engineering Research Organization, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, and the Division of Advanced Therapeutics for Metabolic Diseases, Center for Translational and Advanced Animal Research, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan

Received for publication, December 31, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sortilin has been implicated in the formation of insulin-responsive GLUT4 storage vesicles in adipocytes by regulating sorting events between the trans-Golgi-network and endosomes. We herein show that sortilin serves as a potent myogenic differentiation stimulator for C2C12 myocytes by cooperatively functioning with p75NTR, which subsequently further contributes to development of the insulin-responsive glucose transport system in C2C12 myotubes. Sortilin expression was up-regulated upon C2C12 differentiation, and overexpression of sortilin in C2C12 cells significantly stimulated myogenic differentiation, a response that was completely abolished by either anti-p75NTR- or anti-nerve growth factor (NGF)-neutralizing antibodies. Importantly, small interference RNA-mediated suppression of endogenous sortilin significantly inhibited C2C12 differentiation, indicating the physiological significance of sortilin expression in the process of myogenesis. Although sortilin overexpression in C2C12 myotubes improved insulin-induced 2-deoxyglucose uptake, as previously reported, this effect apparently resulted from a decrease in the cellular content of GLUT1 and an increase in GLUT4 via differentiation-dependent alterations at both the gene transcriptional and the post-translational level. In addition, cellular contents of Ubc9 and SUMO-modified proteins appeared to be increased by sortilin overexpression. Taken together, these data demonstrate that sortilin is involved not only in development of the insulin-responsive glucose transport system in myocytes, but is also directly involved in muscle differentiation via modulation of proNGF-p75NTR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
One of the major physiological roles of insulin is the control of postprandial blood glucose levels, and skeletal muscle is the primary tissue responsible for the bulk (70-80%) of insulin-stimulated postprandial glucose disposal (1, 2). The effect of insulin on overall glucose disposal is primarily achieved via stimulation of glucose uptake into insulin-target tissues, and defects in this insulin action in skeletal muscle contribute to development of the insulin resistance which is characteristic of type 2 diabetes (3).

In skeletal muscle, glucose transport is regulated by a facilitative glucose transport system involving at least two members of the glucose transporter family, GLUT1 and GLUT4 (4), and their expression levels are strictly regulated during myocyte differentiation (5, 6). GLUT1 is targeted predominantly to the sarcolemma (muscle plasma membrane) and is therefore implicated in the regulation of basal glucose transport, although a marked reduction in GLUT1 expression occurs during muscle differentiation (5, 6). In contrast, expression of the insulin-responsive glucose transporter GLUT4 is remarkably up-regulated upon muscle differentiation, and GLUT4 protein is predominantly localized in intracellular tubulo-vesicular elements under basal conditions (7, 8), while insulin stimulates the translocation of GLUT4 from intracellular storage compartments to the cell surface, resulting in the insulin-responsive augmentation of glucose uptake (9). Thus, differentiation-dependent changes in the amount and composition of these GLUT proteins, especially the increase in cellular GLUT4 content, contribute to development of the insulin-responsive glucose transport system in myocytes and adipocytes (5, 10). However, GLUT4 expression is not the only factor responsible for conferring the insulin-stimulated glucose uptake function in these insulin-responsive cells, because ectopic expression of GLUT4 in other cell types such as fibroblasts is insufficient to generate an insulin-stimulated glucose transport system (11, 12).

Recent studies using adipocytes have revealed that one of the additional factors prerequisite for higher insulin responsiveness is a signaling cascade developed during the course of adipocyte differentiation that is absent in pre-adipocytes (13, 14). In addition to differentiation-dependent establishment of this signaling cascade, Kandror and colleagues (15, 16) recently reported that sortilin, a type I transmembrane glycoprotein, which is implicated in the sorting of Vps10p-interacting proteins between the trans-Golgi network, plasma membrane, and lysosomes, serves as another important factor that promotes development of the insulin-induced glucose transport system by producing insulin-responsive GLUT4 storage vesicles. They found that fibroblasts ectopically expressing both sortilin and GLUT4 display significantly augmented glucose uptake in response to insulin stimulation (12). Although it has already been reported that sortilin is expressed in muscle (17, 18), at present the functional role of sortilin in muscle is entirely unknown, although muscle is the tissue in which the insulin effect on postprandial glucose disposal is quantitatively most important.

Sortilin was originally purified from human brain extracts using receptor-associated protein affinity chromatography (17) and was also identified as a major component of GLUT4-containing vesicles from rat adipocytes (18, 19). Sortilin has a Vps10p domain in its luminal region at the N terminus and therefore belongs to the mammalian VPS10p family of sorting receptors. The intraluminal domain of sortilin was shown to interact with a wide array of proteins, including an unprocessed form of nerve growth factor (proNGF),3 neurotensin, lipoprotein lipase, precursor of brain-derived neurotrophic factor (BDNF), and prosaposin, as well as receptor-associated protein (16, 17, 20-23). Therefore, sortilin functions not only to direct the intracellular movements of such newly synthesized interacting proteins but also functions as a receptor for these proteins once sortilin is exposed to the cell surface plasma membrane. Nevertheless, the cytoplasmic tail of sortilin does not possess a domain enabling it to directly activate the intracellular signaling cascade (24). Recent studies have, however, revealed that sortilin forms a complex with proNGF and p75NTR, a low affinity NGF receptor, and triggers p75NTR signaling cascades leading to various cellular responses, including differentiation, survival, and apoptosis (22, 25). Interestingly, genes encoding precursors of NGF and BDNF, and their common low affinity receptor p75NTR, were shown to be expressed in C2C12 myoblasts (26). In addition, several lines of evidence indicate that NGF affects myogenic differentiation and muscle development in an autocrine fashion via p75NTR (26-28).

In the present study, we explored the functional roles of sortilin in muscle by using the skeletal muscle cell line C2C12. We herein report sortilin to be a potent differentiation stimulator functioning cooperatively with p75NTR, which subsequently further contributes to development of the insulin responsive glucose transport system in C2C12 cells. We present compelling evidence that sortilin is involved not only in generating insulin-responsive GLUT4 storage vesicles but also in elaborating an entire glucose transport system exhibiting much higher insulin responsiveness via regulation of the processes of myogenesis, including expressions of GLUT proteins and perhaps various other proteins involved in the development of insulin responsiveness. Thus, our results provide new insights into the functional roles of sortilin in myogenesis and in development of the insulin-responsive glucose transport system in myocytes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—We obtained 2-deoxy-[3H]glucose (37.2 Ci/mmol) from PerkinElmer Life Sciences. The Western blot detection kit (West super femto detection reagents) was from Pierce. Dulbecco's Modified Eagle Medium (DMEM), penicillin/streptomycin and Trypsin-EDTA were purchased from Sigma. Cell culture equipment was from BD Biosciences (San Jose, CA). Calf serum and fetal bovine serum were obtained from BioWest (Nuaille, France). Immobilon-P was from Millipore Corp. (Bedford, MA). Bovine serum albumin (BSA) was purchased from Wako (Osaka, Japan). Antibodies against sortilin, p115, syntaxin 6, Ubc9, and SUMO-modified protein 1 (GMP1) were purchased from BD Biosciences. Anti-sortilin antibody obtained from Abcam (Cambridge, UK) was used for immunofluorescent analyses. Antibodies against cation-independent mannose 6-phosphate/insulin-like growth factor-2 receptor, insulin receptor β-subunit, and c-Myc (9E10) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-myosin heavy chain (MHC) (MF20), anti-myogenin (F5D), anti-troponin T (CT3), and anti-titin (9D10) antibodies were obtained from Iowa Hybridoma Bank (University of Iowa, Iowa City, IA). Anti-sarcomeric {alpha}-actinin (EA-53) and anti-β-actin (A-2066) antibodies were purchased from Sigma. Anti-GLUT1 antibody was purchased from Chemicon International Inc. (Temecula, CA). Anti-p75NTR antibody (ME20.4) was obtained from Abcam. Anti-GLUT4 antibody was a generous gift from Dr. H. Shibata (Gunma University, Maebashi, Japan) (29). Fluorescence-conjugated secondary antibodies were obtained from Invitrogen. Unless otherwise noted, all chemicals were of the purest grade available from Sigma Chemicals.

Cell Culture—Myoblasts from the mouse skeletal muscle cell line C2C12 (30) were maintained in DMEM supplemented with 10% fetal bovine serum, 30 mg/ml penicillin, and 100 mg/ml streptomycin (growth medium) at 37 °C under a 5% CO2 atmosphere. For biochemical study, cells were grown on 6-well plates (BD Biosciences) at a density of 3 x 104 cells/well in 3 ml of growth medium. Three days after plating, the cells had reached ~80-90% confluence (Day 0). Differentiation was then induced by switching the growth medium to DMEM supplemented with 2% calf serum, 30 µg/ml penicillin, and 100 µg/ml streptomycin (differentiation medium). The differentiation medium was changed every 24 h. For the immunofluorescent staining study, cells were grown on 22-mm glass coverslips (Matsunami C022221, Osaka, Japan) in 6-well plates.

Generation of C2C12 Cells Expressing Sortilin—Human sortilin cDNA was inserted into a pMXs retroviral vector, which is generated by modifying the pBABE retroviral vector (31). The pMXs-sortilin or pMXs (control) was transfected into Plat E cells using FuGENE 6 transfection reagents (Roche Applied Science, Indianapolis, IN), and high titer retroviral supernatants were obtained. C2C12 myoblasts were infected with the generated retrovirus in growth medium containing 10 µg/ml Polybrene. Two days after infection, positive selection was performed in the presence of 5 µg/ml puromycin, and 20 individual clones were isolated. The sortilin expression level in each clone was analyzed by Western blotting as described below.

Adenoviral Expression of Myc-GLUT4-ECFP in C2C12 Cells—An adenoviral technique was used to express rat GLUT4, possessing the c-Myc epitope tag in the first extracellular loop and enhanced cyan fluorescent protein (ECFP) at the C terminus. Briefly, established exofacial-Myc-GLUT4-ECFP cDNA (32) was inserted into adenoviral vector pHMCA5, generously provided by Dr. H. Mizuguchi (33, 34). The high titer viruses were produced by transfection of pHMCA5-Myc-GLUT4-ECFP or empty vector into 293 cells using FuGENE 6 transfection reagents (Roche Applied Science). Viral stocks of 5-50 x 107 plaque-forming units/ml were prepared in serum-free DMEM and frozen at -80 °C. The next day the medium was changed, and incubation was continued for another 24 h. Then, 2-deoxy[3H]glucose and Myc antibody uptake assays were performed as described below. Expression of Myc-GLUT4-ECFP was confirmed by Western blotting and immunofluorescent staining.

Western Blot Analysis—The expression levels of each protein were analyzed by Western blotting. In brief, cells were lysed with NET buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 100 units/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 5 µg/ml pepstatin A). The extracts were centrifuged at 13,000 x g for 10 min at 4 °C to remove insoluble materials. The protein concentrations of supernatants were determined, and 3x Laemmli sample buffer was added to achieve a 1x final concentration. From 50 to 100 µg of total protein was subjected to 7.5-10% SDS-PAGE (1:30, bis:acrylamide). Proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore Corp.), and the membranes were then blocked for 2 h at room temperature with 5% BSA in Tris-buffered saline (TBS) containing 0.1% Tween 20. Immunoblotting to detect each protein was achieved with an overnight incubation at 4 °C with 3% BSA/TBS containing either anti-sortilin antibody (1:1000), anti-myosin heavy chain (MHC) antibody (1:1000), anti-myogenin antibody (1:1000), anti-sarcomeric {alpha}-actinin antibody (1:1000), or anti-troponin T antibody (1:1000). Anti-β-actin (1:500) or anti-insulin receptor β-subunit (1:1000) antibodies were used as a loading control. Specific total proteins were visualized after subsequent incubation with a 1:5000 dilution of anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase and a SuperSignal Chemiluminescence detection procedure (Pierce). Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Pierce). Three independent experiments were performed for each condition.

2-Deoxyglucose Uptake Assay—A 2-deoxy[3H]glucose uptake assay was performed as previously described (35). Briefly, cells were starved in serum-free DMEM for 4 h, and then washed with Krebs-Ringer phosphate HEPES buffer (KRPH buffer; 10 mM phosphate buffer, pH 7.4, 1 mM MgSO4, 1 mM CaCl2, 136 mM NaCl, 4.7 mM KCl, 10 mM HEPES (pH 7.6)). The serum-starved cells were incubated without or with 100 nM insulin for 60 min in KRPH buffer and then chilled on ice. After washing with ice-cold KRPH buffer, glucose transport was determined by addition of 2-deoxy[3H]glucose (PerkinElmer Life Sciences, 0.1 mM, 0.5 µCi/ml). After 4 min of incubation with the KRPH buffer containing 2-deoxy[3H]glucose, the reaction was stopped by adding phosphate-buffered saline (PBS) containing 10 µM cytochalasin B (Sigma), and the cells were washed three times with ice-cold PBS. The cells were then lysed in 0.2 N NaOH solution, and 2-deoxy[3H]glucose uptake was assessed by scintillation counting. 20 µM cytochalasin B were added to the assay buffer for the measurement of nonspecific background. Specific uptake, i.e. the background subtracted from the total uptake, was obtained. The protein content was determined in each experiment with a BCA protein assay kit. Data are presented as picomoles of 2-deoxy[3H]glucose per milligram protein per minute. For each experiment, at least three assays were performed under each condition, and each experiment was repeated at least three times.

Anti-Myc Antibody Uptake Assay—For the measurement of insulin-induced GLUT4 translocation, the C2C12 cells expressing Myc-GLUT4-ECFP were starved in serum-free DMEM for 4 h, washed three times with KRPH buffer, and then placed in a CO2 incubator with 2 ml of KRPH buffer. Ten minutes after incubation, the cells were treated with or without 100 nM insulin in the presence of 4 µg/ml of the anti-Myc antibody for 60 min. Next, the cells were washed three times with ice-cold PBS, harvested using 1x Laemmli sample buffer without bromphenol blue and 2-mercaptoethanol (2-ME). After the protein concentration had been determined, bromphenol blue and 2-mercaptoethanol were added, denatured by incubation for 5 min at 95 °C, and subjected to Western blotting using anti-mouse IgG antibody, anti-Myc antibody, or anti-Sortilin antibody.

Immunofluorescence and Image Analysis—After experimental treatments, the cells were washed in PBS and fixed for 20 min in 2% paraformaldehyde/PBS containing 0.1% Triton X-100. The cells were washed, and then blocked in PBS containing 5% calf serum plus 1% BSA for 1 h at room temperature. Primary and secondary antibodies were used at 1:100 and 1:1000 dilutions, respectively (unless otherwise indicated), in 1% BSA/PBS, and samples were mounted on glass slides with Vectashield (Vector Laboratories, Burlingame, CA). Cells were imaged using a confocal fluorescence microscope (Olympus Fluoview FV-1000) with associated application program ASW Ver. 1.3 (Olympus, Tokyo, Japan). Images were then imported into Adobe Photoshop (Adobe Systems, Inc.) for processing. Myogenin-positive cells were counted within a 500-µm x 500-µm area. At least five fields were counted for each sample under 20x microscopic magnification. For all microscopic analyses, at least three independent experiments were performed for each condition.

Subcellular Fractionation of C2C12 Cells—To detect endogenous GLUT1 and GLUT4, fractionation was performed according to the method of Tortorella and Pilch (36). Briefly, cells (three 150-mm plates for each sample) were washed with PBS three times, harvested with a cell scraper, suspended in 0.25 M sucrose, 20 mM Hepes-HCl (pH 7.4), 1 mM EDTA, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride, then homogenized with a Teflon glass homogenizer. The homogenate was centrifuged at 600 x g for 10 min at 4 °C. The supernatant was centrifuged at 100,000 x g for 1 h to collect the pellet for the total membrane fraction. For detection of GLUT1, the total membrane fraction was lysed in 100 µl of 1x Laemmli sample buffer, and subjected to Western blotting using anti-GLUT1 antibody (1:5840). For detection of GLUT4, the total membrane fraction was lysed in 100 µl of 8 M urea, 5% SDS, and 50 mM Tris-HCl (pH 6.8). After the protein concentration had been determined as described above, dithiothreitol and bromphenol blue were added to each sample (final concentrations, 343.6 µM and 0.005%, respectively). After incubation for 30 min at 37 °C, samples were subjected to SDS-PAGE followed by immunoblotting using anti-GLUT4 antibody (1:500) (37).

Real-time Reverse Transcription-PCR—Total RNA was prepared using TRIzol reagent according to the manufacturer's instructions and was quantified using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Reverse transcription was carried out using the First Strand cDNA Synthesis kit for PCR from Roche Applied Science. Real-time PCR reactions were performed using the Roche LightCycler, utilizing Roche SYBR Green reagents according to the manufacturer's instructions. Amplification of PCR products was quantified during PCR by measuring fluorescence associated with binding of double-stranded DNA to the SYBR Green dye incorporated into the reaction mixture. The sequences of the oligonucleotides used to PCR-amplify the cDNAs of interest were: GLUT4 (upper primer, 5'-TGCTCTCCTGCAGCTGATT-3'; lower primer, 5'-TTCAGCTCAGCTAGTGCGTC-3'), GLUT1 (upper primer, 5'-CTTCCTGCTCATCAATCGT-3'; lower primer, 5'-AGCTCCAAGATGGTGACCTT-3'), sortilin (upper primer, 5'-AATGGTCGAGACTATGTTGTG-3'; lower primer, 5'-CCGGTACCCATTTGTTGT-3'), and Ubc9 (upper primer, 5'-GGCACAATGAACCTGATGAAC-3'; lower primer, 5'-TTGGTGGTGAGGACGGATAGT-3'). Glyceraldehyde-3-phosphate dehydrogenase was quantified as a housekeeping gene by using: upper primer 5'-GGAGAAACCTGCCAAGTATGA-3', and lower primer 5'-GCATCGAAGGTGGAAGAGT-3'. Following an initial denaturation step of 95 °C for 1 min, 30-45 cycles of 95 °C for 10 s, 60 °C for 1 s, and 72 °C for 10 s were used for GLUT4, GLUT1, sortilin, and Ubc9. For glyceraldehyde-3-phosphate dehydrogenase, an initial denaturation step of 95 °C for 10 min, followed by 45 cycles of 95 °C for 10 s, 57.5 °C for 15 s, and 72 °C for 15 s was used.

siRNA-mediated Reduction of Sortilin in C2C12 Cells—The siRNA species purchased from Nippon EGT Co. Ltd. (Toyama, Japan) were designed to target the following cDNA sequences: scrambled, 5'-AGGGUGGGUUUGGCCAAAATT-3'; and sortilin siRNA-1, 5'-GGUGGUGUUAACAGCAGAGTT-3'; sortilin siRNA-2, 5'-CCAUUGGUGUGAAAAUCUA-3', and sortilin siRNA-3, 5'-GGACCACAUUACUAUACCA-3'. Scramble and sortilin siRNA-1 were modified from the reference (38), and the sequences of sortilin siRNA-2 and -3 were provided by Nippon EGT Co. Ltd. 200 nmol of sortilin siRNA, or scrambled siRNA species, was introduced into C2C12 myoblasts using Oligofectamine (Invitrogen). Ninety-six hours after transfection (day 3 of differentiation), cells were harvested with NET buffer followed by SDS-PAGE and immunoblotting using anti-sortilin antibody, anti-myogenin antibody, or anti-β-actin antibody (as a control).

Measurement of GLUT Stability—Adenoviral infected WT- and sort10-C2C12 cells expressing Myc-GLUT4-ECFP were treated with 10 µg/ml cycloheximide, and the cells were then harvested with NET buffer at the indicated time intervals. Cell lysates were subjected to SDS-PAGE, followed by Western blotting analysis using anti-GLUT1 or anti-Myc antibody.

Statistical Analysis—Results are expressed as means ± S.E., and the data were analyzed by analysis of variance followed by Student's t test. Differences were considered to be significant at *, p < 0.05; **, p < 0.01; and ***, p < 0.001.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Sortilin Expression during Differentiation of C2C12 Cells—To investigate the physiological role of sortilin in C2C12 myocytes, we first determined changes in expression levels of sortilin during myogenesis of C2C12 cells by Western blotting (Fig. 1A, upper panel). Sortilin was undetectable in undifferentiated C2C12 myoblasts (Day 0) but was observed as a ~100-kDa protein at Day 2 of differentiation when these myoblasts were differentiated in a differentiation medium containing 5 mM glucose. The amount of sortilin then gradually increased until Day 5 and remained unchanged up to Day 8. As a control for myogenic differentiation, expression levels of myogenin (middle panel) and MHC (lower panel) were also determined. Immunofluorescent staining demonstrated sortilin containing endosomes to be located throughout the cytoplasm. Thus, colocalizations with GLUT4 (panels a and b, red), cation-independent mannose 6-phosphate/insulin-like growth factor-2 receptor (panels c and d, red), and Syntaxin 6 (panels e and f, red) were obvious, although the sortilin-positive endosomes were apparently devoid of p115 (panels g and h, red), a cis-Golgi marker in differentiated C2C12 myotubes.

Expression of Sortilin Induces Spontaneous Myogenic Differentiation of C2C12 Cells—To explore the functional roles of sortilin in C2C12 myocytes, we established C2C12 cells in which sortilin is stably expressed using a retroviral technique. In the process of isolating positive clones, we unexpectedly found that overexpression of sortilin in C2C12 myoblasts often resulted in spontaneous myotubular formation even in growth medium containing 10% fetal bovine serum (data not shown). Twenty clones of drug-resistant C2C12 cells were isolated, nine of which stably expressed sortilin as assessed by Western blotting analysis. All nine clones expressing detectable levels of exogenous sortilin displayed the myotubular phenotype once they reached confluence. Three independent clones expressing sortilin, two control clones expressing empty vector, and parental wild-type cells were further analyzed. As shown in Fig. 2, in growth medium, neither wild-type C2C12 (WT-C2C12) nor the control clone expressing empty vector expressed sortilin (Fig. 2A, right panel, lanes 1 and 2). Furthermore, neither displayed a myotubular phenotype even after reaching confluence (Fig. 2A, panels a and b). In sharp contrast, C2C12 clones expressing exogenous sortilin (Sort10- and Sort15-C2C12) (Fig. 2, right panel, lanes 3 and 4) underwent spontaneous differentiation as evidenced by myotube formation (Fig. 2A, left panels, panels c and d). Immunofluorescent analysis revealed that even before differentiation began Sort10-C2C12 cells already expressed various muscle differentiation markers, including myogenin (Fig. 2B, panel d), MHC (Fig. 2, panel e), and titin (Fig. 2, panel f), observations not made in either WT-C2C12 cells (Fig. 2B, panels a-c) or empty vector-expressing C2C12 cells (Fig. 2B, panels d-f). Essentially the same results were obtained in the other clones overexpressing sortilin, but not in other control C2C12 clone obtained by infection with empty retrovirus lacking an insert, and levels of sortilin protein in the control cells were not significantly different from those observed in the non-infected C2C12 myocytes (data not shown).


Figure 1
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  		FIGURE 1.
Protein expression and subcellular localization of sortilin during C2C12 differentiation. A, C2C12 myoblasts were differentiated in conventional differentiation medium (DMEM containing 5 mM glucose plus 2% calf serum). The differentiation medium was changed every 24 h. Whole cell lysates were obtained daily until day 8 of differentiation. Total protein extracts (22.5 µg/lane) were subjected to SDS-PAGE followed by Western blot analysis using anti-sortilin (upper panel), anti-myogenin (middle panel), and anti-myosin heavy chain (MHC: lower panel) antibodies. B, differentiated C2C12 myotubes (Day 6) were fixed and subjected to immunofluorescent staining using rabbit anti-sortilin antibody (green) with either mouse monoclonal anti-Myc (panels a and b), anti-cation-independent mannose 6-phosphate/insulin-like growth factor-2 receptor (panels c and d), anti-syntaxin 6 (panels e and f), or anti-p115 (panels g and h) antibodies (red). For examining co-localization between sortilin and GLUT4 (panels a and b), Myc-GLUT4-ECFP was transiently expressed by infecting C2C12 cells at Day 4 of differentiation with the adenovirus containing the Myc-GLUT4-ECFP gene and then subjecting these cells to immunofluorescent analysis at 2 days after infection (Day 6 of differentiation). Fluorescence signals were detected under confocal microscopy. The squares indicated in panels a, c, d, and g were magnified and are shown in the right panels. Images are representative fields of view from three independent experiments. Scale bar = 10 µm.

 
This stimulatory effect of sortilin overexpression on myogenesis was further confirmed by Western blot analysis using antibodies against various muscle differentiation marker proteins, including MHC, sarcomeric {alpha}-actinin, troponin T, and myogenin (Fig. 2C). Even at Day 0, Sort10-C2C12 cells already showed considerable elevations of all muscle differentiation marker proteins tested (lane 2), observations not made in WT-C2C12 cells (lane 1). At Day 4 of differentiation, all of these differentiation marker proteins were also detected in WT-C2C12 cells, although their expression levels were obviously lower in these (lane 3) than in Sort10-C2C12 cells (lane 4). At Day 7 of differentiation, most of these expressions reached comparable levels in WT-C2C12 (lane 5) and Sort10-C2C12 (lane 6) cells. Together, these data clearly demonstrate that sortilin overexpression in C2C12 myoblasts results in spontaneous myogenic differentiation once confluence is reached even under growth stimulating conditions.

Involvement of the proNGF-p75NTR Autocrine Loop in the Spontaneous Differentiation of Sortilin-overexpressing C2C12 Cells—Since recent studies have revealed direct interactions among sortilin, p75NTR, and proNGF exerting biological effects in neuronal cells (22, 25), we examined the possible involvement of proNGF and p75NTR, the existence of which in C2C12 cells was previously reported (26, 27), in the process of spontaneous differentiation induced by sortilin overexpression. To address this question, Sort10-C2C12 cells were cultured in the presence or absence of a p75NTR-neutralizing antibody that blocks p75NTR-mediated biological responses (39). The p75NTR-neutralizing antibody significantly reduced the number of Sort10-C2C12 myocytes expressing myogenin in the growth medium, whereas control mouse IgG had no such effect (Fig. 3A, left panel). In addition, essentially the same result was obtained when an NGF-neutralizing antibody that blocks both NGF and proNGF action was added to the culture instead of the p75NTR-neutralizing antibody (Fig. 3A, right panel). We also found the anti-p75NTR antibody to be very efficiently endocytosed only in C2C12 cells expressing sortilin (Fig. 3B, panel d, sortilin; green, mouse IgG; red), not in control cells expressing empty vector (Fig. 3B, panel b). No control mouse IgG was incorporated into C2C12 cells, regardless of sortilin expression during the incubation (Fig. 3B, panels a and c). This observation suggests that sortilin overexpression may facilitate complex formation with p75NTR, leading to efficient incorporation of the anti-p75NTR antibody. These data indicate that the stimulatory effect of sortilin overexpression on myogenesis is dependent on the existence of functional p75NTR that has an ability to form the high-affinity proNGF receptor with sortilin.

Physiological Significance of Endogenous Sortilin Expression in Myogenic Differentiation of C2C12 Cells—Because sortilin expression was markedly up-regulated during C2C12 differentiation (Fig. 1), we examined the physiological significance of sortilin expression in the process of myogenic differentiation using the siRNA-mediated gene silencing technique to decrease the expression of endogenous sortilin (Fig. 3C). Three of the sortilin sequence-specific siRNA oligonucleotides (Oligo #1, #2, and #3) significantly reduced endogenous sortilin expression at Day 2 of C2C12 differentiation (Fig. 3C, upper panel, lanes 1-3), resulting in a concomitant suppression of myogenin expression (middle panel, lanes 1-3), whereas a control scramble oligonucleotide had no effect on the expression of either sortilin or myogenin (lane 4). β-Actin, used as a loading control, was unaffected by siRNA introduction (lower panel). In addition, consistent with the sortilin overexpression experiments (Fig. 3A), either p75NTR- or NGF-neutralizing antibody also significantly inhibited the conventional differentiation process of wild-type C2C12 cells in a low serum differentiation medium (Fig. 3D). These data indicate that endogenous sortilin up-regulated during myogenesis is directly involved in the myogenic differentiation of C2C12 cells, a process possibly mediated through the proNGF-p75NTR autocrine loop.


Figure 2
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  		FIGURE 2.
Sortilin overexpression induces spontaneous myogenic differentiation of C2C12 cells. A, parental (WT, panel a), empty-vector-expressing (panel b) and sortilin-overexpressing (Sort10, panel c; Sort15, panel d) C2C12 cells were grown to confluence (Day 0). Myotubes are indicated by arrowheads. Images are representative fields of view from three independent experiments. Scale bar = 300 µm. Exogenously expressed sortilin in each C2C12 cell culture was detected by Western blotting analysis (right panel). B,WT (panels a-c), empty vector (panels d-f), and Sort10 (panels g-i) C2C12 cells at Day 0 were fixed and subjected to immunofluorescent staining using mouse monoclonal anti-myogenin (panels a, d, and g), anti-MHC (panels b, e, and h), and anti-titin (panels c, f, and i) antibodies, which were visualized with Alexa488-conjugated secondary antibody (green). Nuclei were stained by 4',6-diamidino-2-phenylindole (blue). Images are representative fields of view from three independent experiments. C, whole cell lysates were obtained from WT (lanes 1, 3, and 5) and Sort10 (lanes 2, 4, and 6) C2C12 cells on the indicated days after differentiation and total protein extracts (25 µg/lane) were subjected to Western blot analysis using antibodies against sortilin, MHC, sarcomeric {alpha}-actinin, troponin T, and myogenin. β-Actin was used as a loading control. These are representative immunoblots obtained from three independent experiments.

 
Involvement of ROCK in the Spontaneous Differentiation of Sortilin-overexpressing C2C12 Cells—Since the p75NTR has been shown to activate the small G-protein Rho and its down-stream effector ROCK in various cell types (40, 41), we examined whether this signaling cascade is involved in the spontaneous differentiation of sortilin-overexpressing C2C12 cells (Fig. 3E). The myogenin expression observed in sortilin-overexpressing C2C12 cells at Day 0 was diminished when the cells were cultured for 24 h in the presence of an inhibitor of ROCK, Y27632 (Fig. 3E, lower panel).

Overexpression of Sortilin Contributes to Development of an Insulin-induced Glucose Transport System in C2C12 Cells—To assess whether an insulin-responsive glucose transport system develops in skeletal muscle C2C12 cells under the same conditions as previously reported (12), WT- and Sort10-C2C12 myoblasts expressing Myc-GLUT4-ECFP were obtained using an adenoviral vector, and 2-deoxy[3H]glucose (Fig. 4) and Myc Ab (Fig. 5) uptake assays (35) were then performed. Because Sort10-C2C12 cells tend to undergo spontaneous myotubular formation, we carried out the assay before they reached confluence (Day-1). Nevertheless, some differentiation markers were already being expressed at this time point (data not shown). Under the conventional 2-deoxy[3H]glucose uptake assay protocol (42), insulin-induced augmentation of 2-deoxy[3H]glucose uptake was not observed in either WT-C2C12 myoblasts (Fig. 4A, WT) or WT-C2C12 myoblasts expressing exogenous Myc-GLUT4-ECFP (Fig. 4A, WT+G4). Intriguingly, in Sort10-C2C12 myoblasts, basal 2-deoxy[3H]glucose uptake was remarkably decreased, by ~25% (2.89 ± 0.2 pmol/min/mg of protein) as compared with that of WT-C2C12 myoblasts, whereas Sort10-C2C12 myoblasts failed to display any insulin responsiveness probably due to negligible expression of GLUT4 (Fig. 4A, Sort). However, consistent with a previous study using 3T3 fibroblasts (12), a slight but significant insulin-stimulated 2-deoxy[3H]glucose uptake (~1.5-fold) was observed in Sort10-C2C12 myoblasts expressing Myc-GLUT4-ECFP (Fig. 4A, Sort+G4). This occurred concurrently with a small increase in basal 2-deoxy[3H]glucose uptake.

Effects of sortilin overexpression on insulin-induced glucose uptake were also examined in differentiated C2C12 myotubes (Days 5-6) (Fig. 4B). Because differentiated C2C12 myotubes contain massive amounts of various muscle proteins such as skeletal muscle type myosins and {alpha}-actin, normalization by total protein contents for comparing glucose uptake between myoblasts and myotubes may not directly reflect the actual capacity for glucose transport into individual cells. However, it should be noted that the net amount of 2-deoxy[3H]glucose uptake was remarkably low (1.26 ± 0.1 pmol/min/mg of protein) in differentiated C2C12 myotubes, being nearly 10-fold lower than that in undifferentiated C2C12 myoblasts (Fig. 4, A and B). Consistent with our previous report (35), insulin-induced 2-deoxy[3H]glucose uptake was marginal even in differentiated C2C12 myotubes (Fig. 4B, WT) under the conventional uptake assay protocol. The adenovirus-mediated expression of Myc-GLUT4-ECFP raised 2-deoxy[3H]glucose uptake under both basal and insulin-stimulated conditions, and insulin-responsiveness was slightly improved in differentiated WT-C2C12 myotubes (Fig. 4B, WT+G4). Consistent with the result obtained in undifferentiated myoblasts expressing sortilin (Fig. 4A, Sort), differentiated Sort10-C2C12 myotubes showed a further reduction in net 2-deoxy[3H]glucose uptake (Fig. 4B, Sort). Because of this suppression of basal glucose uptake, a slight but significant augmentation of insulin-induced 2-dexoy[3H]glucose uptake was subsequently seen even without exogenous expression of Myc-GLUT4-ECFP (Fig. 4B, Sort), presumably reflecting glucose uptake mediated through endogenous GLUT4 translocation in differentiated Sort10-C2C12 myotubes. The adenovirus-mediated expression of Myc-GLUT4-ECFP in Sort10-C2C12 myotubes further enhanced insulin-responsiveness (2.1-fold increase in response to insulin), and this was concurrent with a slight increase in basal 2-deoxy[3H]glucose uptake (Fig. 4B, Sort+G4).


Figure 3
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  		FIGURE 3.
Involvement of sortilin-p75NTR-proNGF autocrine loop in the process of C2C12 differentiation. A, WT (open bars) and Sort10 (solid bars) C2C12 cells on Day-1 with the addition of either control IgG, anti-p75NTR-neutralizing antibody (left panel), or anti-NGF-neutralizing antibody (right panel), followed by culture for an additional 24 h. The cells were then fixed and subjected to immunofluorescent staining using mouse monoclonal anti-myogenin visualized with Alexa594-conjugated secondary antibody. Nuclei were stained by 4',6-diamidino-2-phenylindole, and the number of myogenin positive nuclei was counted. The ratio of myogenin-positive nuclei out of total nuclei was expressed as the mean ± S.E. of four independent experiments. *, p < 0.05; **, p < 0.01. B, empty vector (panels a and b) or Sort10 (panels c and d) C2C12 cells seeded in an 8-well slide chamber were treated with 10 µg/ml of anti-p75NTR antibody or mouse IgG before they reached confluence, followed by culture for an additional 24 h. To detect incorporated IgGs, the cells were subjected to immunostaining with anti-mouse IgG (red) and anti-sortilin antibody (green), as described in A). C, WT-C2C12 cells (Day-1) were transfected with siRNA oligonucleotides targeting sortilin (lanes 1-3) or control scrambled siRNA oligonucleotide (lane 4) by Oligofectamine for 24 h and then cultured in differentiation medium for an additional 3 days. Total protein extracts (135 µg/lane) were subjected to Western blot analysis using anti-sortilin (upper panel), anti-myogenin (middle panel), or anti-β-actin (lower panel) antibodies. D, confluent WT-C2C12 cells were cultured in differentiation medium in the absence or presence of 10 µg/ml of either control IgG, anti-p75NTR or anti-NGF antibodies for 48 h. The ratio of myogenin-positive nuclei was assessed by immunostaining analysis as described in A. E, empty vector (upper panel) or Sort10 (lower panel) C2C12 cells were incubated with or without the indicated concentrations of Y27632 for 24 h. The whole cell lysates were then subjected to Western blotting analysis using anti-myogenin antibody.

 
Effect of Sortilin Overexpression on Insulin-induced GLUT4 Translocation in C2C12 Cells—To confirm that insulin-induced 2-deoxy[3H]glucose uptake was in fact achieved through the translocation of GLUT4 to the plasma membrane, the amount of Myc-GLUT4-ECFP exposed to the cell surface during insulin stimulation was assessed using an anti-Myc antibody (Myc Ab) uptake assay (Fig. 5) (35). The serum-starved WT- or Sort10-C2C12 myotubes expressing Myc-GLUT4-ECFP (Days 5-6) were incubated for 1 h in KRPH buffer containing 4 µg/ml Myc Ab in the absence (Fig. 5, lanes 1 and 3) or presence (lanes 2 and 4) of insulin (100 nM), and whole cell lysates were then subjected to SDS-PAGE followed by Western blotting using antibodies against anti-mouse IgG for detecting Myc Ab uptake and anti-BD-living colors for detecting the total amount of Myc-GLUT4-ECFP expressed. Consistent with our previous report (35), a small but significant increase in the uptake of Myc Ab was observed with insulin stimulation in WT-C2C12 myotubes expressing Myc-GLUT4-ECFP (Fig. 5, upper panel, lanes 1 and 2). Importantly, insulin-responsive GLUT4 translocation as assessed by the Myc Ab uptake assay appears to be significantly improved in Sort10-C2C12 myotubes (Fig. 5, lane 4), despite marked suppression of net 2-deoxy[3H]glucose uptake in comparison with WT-C2C12 myotubes (Fig. 4B). Together, these data clearly demonstrate that sortilin overexpression improves insulin-responsive glucose transport by increasing insulin-responsive GLUT4 translocation in C2C12 myotubes. Our data also indicate the importance of decreased basal glucose uptake in the emergence of markedly increased insulin-responsive glucose uptake.

Effects of Sortilin Overexpression on Expressions of GLUT1 and GLUT4 in C2C12 Cells—Because sortilin overexpression markedly decreased basal glucose uptake (Fig. 5) and also appeared to stimulate C2C12 differentiation (Figs. 1 and 2), we examined protein and mRNA expressions of endogenous GLUT1 and GLUT4 in WT- and Sort10-C2C12 cells during myogenesis (Fig. 6). Western blot analysis demonstrated that GLUT1 protein decreased remarkably during differentiation of C2C12 cells (Fig. 6A, left panel, lanes 1, 3, and 5). Consistent with our finding that sortilin overexpression suppresses basal glucose uptake (Fig. 4), an obvious reduction of GLUT1 protein was observed in Sort10-C2C12 myoblasts (Fig. 6A, left panel, lane 2) as compared with that in WT-C2C12 myoblasts (lane 1). Upon differentiation, Sort10-C2C12 myotubes displayed marginal levels of GLUT1 protein (Fig. 6A, left panel, lanes 6 and 7), resulting in lower basal 2-deoxy[3H]glucose uptake than that observed in WT-C2C12 myotubes (Fig. 4B). Real-time PCR analysis using a LightCycler (Roche Applied Science) revealed that GLUT1 mRNA expression was significantly decreased in Sort10-C2C12 cells as compared with WT-C2C12 cells even at Day 0 (Fig. 6B). Upon differentiation, expression of GLUT1 mRNA reached minimum levels and was present in comparable amounts in WT- and Sort10-C2C12 myotubes on Days 4 and 7 (Fig. 6B). We have also confirmed that sortilin overexpression resulted in increased expression of GLUT4 as assessed by both Western blotting (Fig. 6A, right panel) and real-time PCR analysis (Fig. 6B, right graph). Because decreased GLUT1 and increased GLUT4 expression are important criteria for myogenesis (43, 44), these data further confirm that sortilin functions as a potent differentiation stimulator in C2C12 cells. It is also noteworthy that, despite the similar expression levels of GLUT1 mRNA in differentiated WT- and Sort10-C2C12 myotubes (Fig. 6B, Days 4 and 7), cellular contents of GLUT1 protein were significantly reduced in Sort10-C2C12 (Fig. 6A, lanes 4 and 6) in comparison with WT-C2C12 (Fig. 6A, lanes 3 and 5) myotubes.


Figure 4
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  		FIGURE 4.
Effects of sortilin overexpression on basal and insulin-stimulated 2-deoxy[3H]glucose uptake in C2C12 cells. A, undifferentiated WT (open bars, WT) and Sort10 (solid bars, Sort) C2C12 myoblasts were infected with adenovirus containing the Myc-GLUT4-ECFP gene (G4) or no insert. Thirty-six hours after infection, the cells were serum starved for 4 h, followed by preincubation with KRPH buffer for 10 min, and then stimulated with or without 100 nM insulin for 60 min. The cells were then subjected to the 2-deoxy[3H]glucose uptake assay as described under "Experimental Procedures." Results were expressed as the mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01. B, WT (open bars, WT) and Sort10 (solid bars, Sort) C2C12 myotubes at Day 5 of differentiation were infected with adenovirus containing the Myc-GLUT4-ECFP gene (G4) or no insert. Thirty-six hours after infection, the cells were subjected to the 2-deoxy[3H]glucose uptake assay as described in A. Results were expressed as the mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01.

 


Figure 5
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  		FIGURE 5.
Effect of sortilin overexpression on insulin-induced GLUT4 translocation in C2C12 cells. A, differentiated WT (open bars) and Sort10 (solid bars) C2C12 myotubes at Day 5 were infected with adenovirus containing the Myc-GLUT4-ECFP gene. Thirty-six hours after infection, the cells were serum starved for 4 h, followed by stimulation with or without 100 nM insulin for 60 min. During the insulin stimulation, 4 µg of anti-Myc antibody was added to the culture. After insulin stimulation, amounts of associated/internalized antibody (upper panel) were analyzed by Western blotting. The total amount of Myc-GLUT4-ECFP (lower panel) was also analyzed by Western blotting for normalization. B, the results of three independent experiments were quantified using ImageJ software. Summarized results were expressed as the mean ± S.E.

 
To assess whether sortilin overexpression alters the stability of GLUT proteins, we next examined time-dependent changes in cellular contents of GLUT1 and GLUT4 in the presence of cycloheximide (Fig. 6C). Consistent with a previous report (12), GLUT4 protein displayed greater stability in sortilin-overexpressing C2C12 myoblasts (right panels). On the other hand, we found that GLUT1 tended to be less stable in sortilin-overexpressing cells (left panel). Taken together, these data indicate that, in addition to the translational regulation, post-transcriptional regulation of GLUT1 protein is also altered by sortilin overexpression.

Increased Ubc9 Expression in Sort10-C2C12 Cells—Cellular contents of GLUT1 and GLUT4 are reportedly regulated post-transcriptionally by exogenous Ubc9 expression in the L6 skeletal muscle cell line (45). We therefore examined expression levels of Ubc9 by Western blotting (Fig. 7A) and real-time PCR analysis (Fig. 7B) in WT- and Sort10-C2C12 cells during myogenesis. We found that the amount of Ubc9 protein was significantly increased in Sort10-C2C12 cells (Fig. 7A, lanes 2, 4, and 6) as compared with WT-C2C12 cells (lanes 1, 3, and 5) at all differentiation time points examined. Consistent with this, a slight increase in Ubc9 mRNA expression was also observed in Sort10-C2C12 cells (Fig. 7B, closed bars) as compared with WT-C2C12 cells (open bars), whereas expression levels of Ubc9 mRNA decreased upon differentiation in both WT- and Sort10-C2C12 cells. As a result of the increased Ubc9 in Sort10-C2C12 cells, SUMO-modified proteins were also increased (Fig. 7A, right panel, lanes 2, 4, and 6).


Figure 6
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  		FIGURE 6.
Effect of sortilin overexpression on the protein and mRNA levels of GLUT1 and GLUT4 in C2C12 cells. A, total membrane fractions were obtained from WT-C2C12 cells (lanes 1, 3, and 5) and Sort10-C2C12 cells (lanes 2, 4, and 6) at Day 0 (lanes 1 and 2), Day 4 (lanes 3 and 4), and Day 7 (lanes 5 and 6) of differentiation, as described under "Experimental Procedures." Protein extracts from total membranes (120 µg/lane) were subjected to SDS-PAGE followed by Western blotting using antibodies against GLUT1 (left panel) or GLUT4 (right panel). B, total RNA was isolated from WT-C2C12 cells (open bars) and Sort10-C2C12 cells (solid bars) on the indicated day after differentiation. The relative abundances of mRNAs for GLUT1 (left graph) and GLUT4 (right graph) were evaluated by real-time PCR analysis. Data normalized using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript were averaged over three independent experiments and are shown as -fold changes over Day 0 in WT-C2C12 cells. C, time dependent changes in protein amounts of GLUT1 (left panels) and Myc-GLUT4-ECFP (right panels) after addition of 10 µg/ml cycloheximide were monitored in WT (open circles) and Sort10 (closed circles) C2C12 cells by Western blotting.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although substantial progress has been made in our understanding of the insulin-induced GLUT4 translocation process in adipocytes, achieved mainly using the excellent 3T3L1 adipogenic cell culture model (46), much less information is available about the mechanistic details involved in this event in skeletal muscle, the tissue in which the effect of insulin on glucose disposal is quantitatively most important. Recently, we have reported that, much like the L6 skeletal muscle cell line, a C2C12 myogenic cell line derived from mouse skeletal muscle possesses the basic machinery required for GLUT4 translocation in response to insulin stimulation (43, 47). However, the insulin-induced glucose uptake achieved by this GLUT4 translocation is masked by relatively high basal glucose transport activity, presumably mediated through GLUT1 (35). Because it would be highly desirable to establish cell culture models of skeletal muscle that clearly and accurately reflect muscle glucose disposal in vivo, we have attempted to further characterize the molecular details underlying development of the insulin-induced glucose transport system in these C2C12 cells. We therefore explored the functional roles of sortilin, a sorting receptor implicated in the formation of insulin-responsive GLUT4 vesicles in adipocytes (12, 48), by using C2C12 myocytes.

A key finding of the present studies is that sortilin functions as a potent differentiation regulator for C2C12 skeletal muscle cells, at least in part by modulating the p75NTR-proNGF autocrine loop (Figs. 2 and 3). As previously demonstrated in adipocytes (12), sortilin overexpression apparently improves the insulin-induced glucose uptake in C2C12 myocytes (Figs. 4 and 5), a novel observation that, however, reveals that this effect of sortilin on the development of insulin-responsiveness in C2C12 cells reflects a combination of consequences of its myogenic stimulatory action. Indeed, sortilin overexpression significantly stimulates C2C12 differentiation (Fig. 2) and thereby raises the cellular content of GLUT4 and decreases that of GLUT1 by altering both transcriptional and post-transcriptional levels (Fig. 6). Importantly, the differentiation-dependent expression of endogenous sortilin (Fig. 1) appears to be directly involved in the process of myogenesis, because siRNA-mediated suppression of sortilin significantly inhibited C2C12 differentiation (Fig. 3C), which is at least in part mediated through the p75NTR-NGF autocrine loop (Fig. 3D). In addition, we found that cellular contents of Ubc9 and SUMO-modified proteins are remarkably increased by sortilin overexpression (Fig. 7). Although it is not clear from the present study whether the up-regulated Ubc9 is directly responsible for post-translational regulation of GLUT proteins as previously reported (45), we observed that GLUT4 became more stable, whereas GLUT1 became less stable, in sortilin-overexpressing C2C12 cells (Fig. 6C).


Figure 7
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  		FIGURE 7.
Sortilin overexpression increases Ubc9 resulting in the accumulation of SUMO-modified proteins in C2C12 cells. A, whole cell lysates were obtained from WT-C2C12 cells (lanes 1, 3, and 5) and Sort10-C2C12 cells (lanes 2, 4, and 6) at Day 0 (lanes 1 and 2), Day 4 (lanes 3 and 4), and Day 7 (lanes 5 and 6) of differentiation. Protein extracts (25 µg/lane) were subjected to SDS-PAGE followed by Western blotting using antibodies against anti-Ubc9 (upper panel), anti-GMP1 (SUMO-1) (middle panel), or anti-β-actin (lower panel). B, total RNA was isolated from WT-C2C12 cells (open bars) and Sort10-C2C12 cells (solid bars) on the indicated day after differentiation. The relative abundance of mRNAs for Ubc9 was evaluated by real-time PCR analysis. Data normalized using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript were averaged over three independent experiments and are shown as -fold changes over Day 0 in WT-C2C12 cells.

 
Sortilin Serves as a Stimulator of C2C12 Myogenic Differentiation via Activation of the p75NTR-proNGF Autocrine Loop—In cultured myoblasts, including mouse C2C12 cells and rat L6 cells, serum withdrawal initiates the myogenic differentiation program, and it is generally accepted that autocrine/paracrine actions of insulin-like growth factors in response to serum withdrawal play an important role in the process of muscle differentiation (49, 50). In addition to the insulin-like growth factor-mediated autocrine system, several lines of evidence have demonstrated that NGF, and presumably its unprocessed form termed proNGF, is also involved in the initiation of muscle differentiation in an autocrine fashion (26, 27, 51). Furthermore, overexpression of p75NTR, a low affinity receptor for various neurotrophins, including proNGF, proBDNF, NGF, and BDNF, has been shown to induce spontaneous myogenic differentiation in a growth medium (26), which is similar to what we have observed in C2C12 myoblasts overexpressing sortilin (Fig. 2). Because p75NTR appeared to form a complex with sortilin creating high affinity receptors for proNGF and thereby evoking an intracellular signaling cascade (22), we hypothesized that sortilin overexpression allows endogenous p75NTR to respond to limiting concentrations of neurotrophins being secreted by C2C12 cells in an autocrine manner and/or fed with medium containing fetal bovine serum. Consistent with this idea, neutralizing antibodies against either p75NTR or NGF significantly inhibited the spontaneous differentiation of Sort10-C2C12 cells overexpressing sortilin (Fig. 3A). In addition, sortilin overexpression resulted in an efficient endocytosis of the anti-p75NTR antibody from culture media (Fig. 3B), suggesting that exogenously expressed sortilin facilitates formation of the functional tripartite complex composed of proNGF, p75NTR, and sortilin, as previously reported (22). More importantly, either siRNA-mediated suppression of endogenous sortilin expression or specific antibody-mediated neutralization of endogenous p75NTR/proNGF significantly inhibits low serum-induced C2C12 differentiation (Fig. 3, C and D). Together, these data demonstrate that sortilin serves as a stimulator of C2C12 myogenic differentiation mediated through potentiation of the p75NTR-proNGF autocrine loop, and that differentiation-dependent increases in endogenous sortilin participate directly in the promotion of myogenesis. It remains to be clarified whether sortilin has a similar capability in the process of adipocyte differentiation, which is currently under investigation.

It has been reported that sortilin binds mature NGF with low affinity (Kd = 10-8M) and proNGF with high affinity (Kd = 10-9 M), and that the latter affinity is further increased (Kd = 10-10 M) when sortilin associates with p75NTR (22). Therefore, our data raise the novel possibility that levels of sortilin expression regulate myogenic differentiation by modulating the biological actions mediated through the p75NTR-proNGF autocrine loop. Because sortilin expression is up-regulated (Fig. 1), whereas p75NTR and NGF expressions are down-regulated (26), upon differentiation, fine-tuning of the expression levels of these proteins would be required for proper development and maintenance of healthy muscle tissues (28). Although the signaling cascades involved in its myogenic actions have yet to be clarified, several lines of evidence indicate a signaling relationship between p75NTR and the Rho family of small GTP-binding proteins in the process of cellular differentiation (40, 41). In this regard, regulation of Rho family members has been directly implicated in myogenesis (52, 53), and the inhibition of ROCK by either Y29632 or siRNA-mediated gene silencing reportedly potentiated myogenesis of C2C12 cells (54-56). In the present study, however, we found that Y29632 significantly inhibited the myogenin expression observed in sortilin-overexpressing C2C12 cells early in differentiation (Day 0) (Fig. 3E). Since fine-tuning of both Rho family members and ROCK has been shown to be important for myogenesis (54-56), the precise molecular mechanisms underlying the phenomena mentioned above remain to be clarified. However, our results strongly suggest an involvement of ROCK in the potentiation of myogenesis induced by sortilin overexpression.

Effects of Sortilin Expression on the Insulin-responsive Glucose Transport System in C2C12—Sortilin is expressed in a number of tissues, including the brain, testis, and fat, as well as in skeletal muscle and the heart (17, 18, 57). Sortilin has been shown to play an important role in trans-Golgi network-to-endosome and trans-Golgi network-to-lysosome trafficking events (15). Like cation-independent mannose 6-phosphate/insulin-like growth factor-2 receptor, a well known sorting receptor that traffics among the trans-Golgi network, lysosomes, and the plasma membrane, sortilin possesses a short cytoplasmic tail at the C-terminal containing an acidic cluster-dileucine motif that is specifically recognized by the recently identified adaptor proteins termed GGAs (15, 58). In the context of GLUT4 regulation in adipocytes, sortilin expression is up-regulated during 3T3L1 adipocyte differentiation (18, 19), and recent studies by Kandror and colleagues demonstrated a crucial role of sortilin in the formation of insulin-sensitive GLUT4 storage vesicles responsible for the insulin-stimulated glucose transport in 3T3L1 adipocytes (12, 48). Although the precise molecular mechanism involved in the formation of insulin-responsive GLUT4 storage compartments remains largely unknown, the available evidence indicates that sortilin induces the biogenesis of vesicles containing GLUT4 in concert with GGA adaptor proteins in cultured adipocytes (59, 60). More recently, the importance of luminal interactions between sortilin and GLUT4 in endosomes for producing insulin-responsive GLUT4 storage vesicles has been demonstrated (48).

Similar to observations in the 3T3L1 adipogenic cell line (18, 19), our results demonstrate that sortilin is up-regulated upon C2C12 differentiation but is only partially colocalized to GLUT4-containing endosomes (Fig. 1). It should be noted, however, that subcellular organelles underwent a pro-found reorganization during myogenic differentiation, and their localization patterns and architectures differed remarkably between undifferentiated mononuclear myoblasts and differentiated multinuclear myotubes (8, 61). However, the amount of endogenous sortilin seems to be insufficient for conferring any apparent insulin-responsiveness in terms of glucose uptake in C2C12 myotubes, because insulin stimulation of 2-deoxy[3H]glucose uptake was marginal (Fig. 4B), due to a relatively high level of basal glucose uptake mediated through GLUT1. Consistent with previous studies using fibroblastic cell types (12, 48), overexpression of sortilin in myocytes apparently produced insulin-responsiveness as assessed by both the 2-deoxy[3H]glucose uptake assay (Fig. 4) and the Myc Ab uptake assay (Fig. 5). These results support the suggestion of Kandror and colleagues that sortilin plays an essential role in the development of GLUT4 storage vesicles and responsiveness to insulin (12, 48).

However, our aforementioned novel observation that sortilin overexpression significantly potentiates myogenesis sets the stage for an alternative hypothesis that sortilin participates in development of the entire insulin-responsive glucose transport system, being involved in more than just GLUT4 storage compartments, and that this is achieved, at least in part, via its myogenic stimulatory actions. This hypothesis is supported by evidence that sortilin overexpression strongly stimulates expression of the GLUT4 gene while inhibiting that of the GLUT1 gene, leading to opposite changes in GLUT4 and GLUT1 abundance (Fig. 6), which thereby produces lower basal glucose uptake and greater augmentation of glucose uptake in response to insulin (Fig. 4). In addition, emergence of the insulin-responsive glucose uptake in skeletal muscle cells by sortilin overexpression appeared to have resulted, at least in part, from the marked reduction in basal glucose uptake (Fig. 4) due to significantly reduced GLUT1 contents (Fig. 6). In this regard, we observed the half-lives of GLUT proteins to be reciprocally regulated in sortilin-overexpressing C2C12 cells (Fig. 6C). Thus, these data indicate sortilin to be involved not only in generating insulin-responsive GLUT4 storage vesicles, but also in elaborating the entire glucose transport system exhibiting enhanced insulin-responsiveness via regulation of the processes of myogenesis including expressions of GLUT proteins and also perhaps various other proteins involved in the development of insulin-responsiveness.

Another interesting observation presented in this study is that sortilin overexpression resulted in significant increases in the contents of Ubc9 and SUMO-modified proteins in C2C12 cells (Fig. 7). Ubc9 is the only protein serving as an E2-type SUMO-conjugating enzyme in vertebrates, and most of the well known Ubc9 interacting proteins are nuclear or are translocated to the nucleus (62, 63). Intriguingly, however, Ubc9 has been shown to interact directly with GLUT1 and GLUT4 and to modulate their membrane expression levels in opposite directions through a posttranslational mechanism (45). Namely, overexpression of Ubc9 in the L6 skeletal muscle cell line results in a marked decrease in the cellular GLUT1 content and an increase in the GLUT4 content (45), which is in excellent agreement with our present results, shown in Figs. 6 and 7. Thus, the effects of sortilin overexpression on the significant reduction in cellular GLUT1 content, despite similar expression levels of its gene (Fig. 6, A and B, Days 4 and 7), may have resulted from the post-translational regulation of GLUT1 governed by the accumulated Ubc9 (Fig. 7). Although possible involvement of the accumulated Ubc9 in the increase in GLUT4 protein is not clear at this time, because it coincides with up-regulation of GLUT4 gene expression in response to sortilin overexpression (Fig. 6, A and B, right panels), a recent report demonstrated that Ubc9 overexpression results in the inhibition of GLUT4 degradation and promotes its targeting to insulin-responsive GLUT4 storage compartments (64). However, another recent report demonstrated that Ubc9 is also directly involved in the myogenic differentiation of C12C12 cells (65). Thus, Ubc9 seems to have dual functions, and our observations suggest that the sortilin-induced opposite changes in GLUT4 and GLUT1 abundance might reflect a combination of the consequences of the accumulation of Ubc9, which may regulate the half-lives of GLUT proteins and also further participate in the potentiation of myogenesis. Although the degree to which sortilin actions on myogenesis or the opposite changes in GLUT1 and GLUT4 abundance, possibly mediated through Ubc9 accumulation, contributes to enhanced insulin-responsiveness is an important question warranting further research, our findings clearly provide novel insights into the functional roles of sortilin in development of the insulin-responsive glucose uptake system in muscle cells.


   FOOTNOTES
 
* This work was supported in part by grants from the New Energy and Industrial Technology Development Organization and the Ministry of Education, Science, Sports and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. Back

2 Supported by Special Coordinated Funds for Promoting Science and Technology. To whom correspondence should be addressed: Tel.: 81-22-717-7581; Fax: 81-22-717-7578; E-mail: kanzakimakoto{at}mac.com.

3 The abbreviations used are: proNGF, unprocessed form of nerve growth factor; NGF, nerve growth factor; p75NTR, p75 neurotrophin receptor; Vps, vacuolar protein sorting; GGA, Golgi-localizing {gamma}-adaptin ear homology domain, ARF-binding proteins; ARF, ADP-ribosylation factor; siRNA, small interfering RNA; SUMO, small ubiquitin-related modifier; BDNF, brain-derived neurotrophic factor; MHC, myosin heavy chain; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; ECFP, enhanced cyan fluorescent protein; PBS, phosphate-buffered saline; Ab, antibody. Back


   ACKNOWLEDGMENTS
 
We thank Dr. H. Shibata (Gunma University) for providing anti-GLUT4 antibody and Dr. H. Mizuguchi (National Institute of Biomedical Innovation) for providing the pHMCA5 plasmid. We also thank Natsumi Emoto and Fumie Wagatsuma for excellent technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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J. Shi, G. Huang, and K. V. Kandror
Self-assembly of Glut4 Storage Vesicles during Differentiation of 3T3-L1 Adipocytes
J. Biol. Chem., October 31, 2008; 283(44): 30311 - 30321.
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