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lysosomal system and glucose transporter 4
Neuromuscular Disorders 13 (2003) 49–54 www.elsevier.com/locate/nmd
Effect of acid maltase deficiency on the endosomal/lysosomal system and glucose transporter 4 M. Orth a,b,*, R.R. Mundegar c a
b
University Department of Clinical Neuroscience, Royal Free and University College Medical School, London, UK University Department of Clinical and Experimental Epilepsy, Institute of Neurology, University College London, Box 19, Queen Square, London WC1N 3BG, UK c Institut fu¨r Physiologie II, University of Bonn, Wilhelmstr. 31, D-53111 Bonn, Germany Received 18 March 2002; received in revised form 4 July 2002; accepted 17 July 2002
Abstract Membrane bound glycogen storage in muscle is characteristic for the lysosomal storage disorder acid maltase (acid a-glucosidase) deficiency while in phosphofructokinase and phosphorylase deficiency, glycogen is stored free in the cytoplasm. Using immunohistochemistry, we examined whether acid maltase deficiency had an effect on early endosomes, recycling endosomes and trans-Golgi network, vesicle systems linked to lysosomes. Vacuolated glycogen containing fibres stained intensely for the lysosomal marker lysosomal-membrane-protein-1 within fibres and at the sarcolemma. There was a similar increase in immunoreactivity for markers of early endosomes (rab5), recycling endosomes (transferrin receptor) and the trans-Golgi network. In acid maltase deficiency, but not in normal muscle or other glycogenoses, staining for the insulin responsive glucose transporter 4 was markedly increased and partially co-localised with all vesicular markers. Our results suggest an effect of acid maltase deficiency extending to various vesicle systems linked to lysosomes. The enzyme defect may also affect the homoeostasis of receptors cycling through these organelles such as glucose transporter 4. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Vacuolar myopathy; GLUT4; Acid maltase; Glycogen storage; Endosome; Lysosome; Trans-Golgi network
1. Introduction Phosphorylase deficiency, phosphofructokinase (PFK) deficiency and acid maltase (acid a-glucosidase) deficiency (AMD) are associated with glycogen accumulation in skeletal muscle. Phosphorylase and PFK play well-defined roles in glycogenolysis and glycolysis, and their deficiency affects energy production pathways. Glycogen is stored non-membrane bound within the cytoplasm of phosphorylase or PFK deficient muscle. AMD is a lysosomal storage disorder. The role of acid maltase in skeletal muscle is not completely understood. However, the enzyme is thought to be able to degrade glycogen during its metabolic turnover within lysosomes. Hence, in AMD glycogen accumulates within lysosomes. In muscle and adipose cells, glucose and glycogen metabolism are linked to glucose transporter 4 (GLUT4). Glucose uptake increases both with exercise independent of insulin [1] and when insulin triggers the translocation of GLUT4
* Corresponding author. Tel.: 144-207-8373611, ext. 4272; fax: 144207-278-8772. E-mail address: [email protected] (M. Orth).
from cytoplasmic vesicle pools to the plasma membrane [2–4]. Glucose can then bind to GLUT4 and the receptorligand complex is taken up into the cell by endocytosis. There is evidence to suggest that the receptor separates from its substrate in early endosomes and is passed on to recycling endosomes and, subsequently, stored in the GLUT4-containing vesicular compartment [5]. In adipocytes and myoblasts, this compartment has been shown to express proteins characteristic for the trans-Golgi network [6,7], endosomes, and recycling endosomes [8,9]. Intracellular glycogen contents possibly down-regulate GLUT4 expression and thus contribute to GLUT4 homoeostasis [23]. To our knowledge, there are no reports on the expression of GLUT4 in skeletal muscle of patients with glucose and glycogen utilisation disorders. In these patients, an altered GLUT4 expression could help circumvent metabolic blocks and maintain energy requirements or reflect abnormalities in the endosomal/lysosomal pathway. We compared by immunohistochemistry GLUT4 localisation in normal adult skeletal muscle with that from patients with deficiencies of phosphorylase, PFK or AMD, respectively. We correlated expression of GLUT4 with proteins typically found in the membranes of early endosomes
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(rab5), recycling endosomes (transferrin receptor, TfR), late endosomes and lysosomes (lysosomal associated membrane protein-1), and a marker for the trans-Golgi network. 2. Material and methods 2.1. Muscle biopsies Muscle biopsies were examined from five patients with adult-onset AMD, two patients with phosphorylase deficiency, one patient with PFK deficiency, and two normal controls. Diagnosis was based on clinical examination, electrophysiology and muscle biopsies applying routine histological and histochemical techniques [12,13]. The diagnoses of phosphorylase, PFK and AMD deficiency were confirmed biochemically using standard methods. 2.2. Antibodies and immunohistochemistry The following primary antibodies were used: anti-lysosomal associated membrane protein-1 (PharMingen, San Diego, USA, dilution 1/200), anti-TfR (Chemicon, Temecula, USA, 1/5), anti-Golgi zone (Chemicon, 1/200), antirab5 (Transduction Laboratories, Lexington, USA, 1/20), anti-GLUT4 (Chemicon, 1/2000). With the exception of anti-GLUT4, which is a rabbit polyclonal antibody, all antibodies are mouse monoclonal. Seven micrometer transverse sections of muscle biopsies, snap-frozen in liquid nitrogen cooled isopentane, were collected on cover slips coated with 3-aminopropyltriethoxysilan and fixed in acetone for 1 min at 2208C. Blocking of unspecific binding sites with 10% normal horse serum in phosphate-buffered saline (PBS) for 20 min was followed by incubation with the primary antibodies at room temperature for 24 h in a humid chamber. After three washes in PBS, mouse monoclonal antibodies were incubated with biotinylated horse anti-mouse secondary antibodies (Vector Laboratories, Burlingame, USA). Polyclonal rabbit antibodies were developed with biotinylated horse anti-rabbit secondary antibodies (Vector Laboratories). After another three washes in PBS, the sections were incubated with an avidin–biotin complex followed by developing for 5 min with 0.5 mg/ml 3,3 0 diaminobenzidine in PBS and 1 ml/ml H2O2 using the Vectastain-Kit (Vector Laboratories). For fluorescence staining, sections were blocked with 10% normal goat serum. Antibodies raised in mouse were developed for 1 h with Alexa 488 goat anti-mouse IgG conjugate (Molecular Probes, Eugene, USA, 1/200), while the rabbit antibody was detected with Alexa 568 goat antirabbit IgG conjugate (Molecular Probes, 1/1000). Sections were analysed and photographed using a Zeiss Fluorescence Axioplan microscope and a Zeiss MC 80 photocamera. For confocal microscopy, slides were evaluated with a Krypton–Argon laser (BioRad MRC 600) attached to an Olympus BH2-RFCA fluorescence microscope.
Specificity controls consisted of substitution of the primary antibody by irrelevant monoclonal antibodies of the same isotype and species and in the same concentration as the monoclonal primary antibodies, and of replacement of polyclonal antibodies by non-immune serum.
3. Results Muscle sections of patients with AMD, phosphorylase or PFK deficiency, and normal controls, were stained with haematoxylin/eosin (HE) to determine morphology, periodic-acid Schiff (PAS) to assess glycogen content and acid phosphatase to examine lysosomal activity. In AMD, 40–60% of muscle fibres were vacuolated (Fig. 1A, D), showed increased glycogen storage (Fig. 1B, E) and marked acid phosphatase activity (Fig. 1C). In phosphorylase or PFK deficiency, glycogen accumulated in small subsarcolemmal blebs (Fig. 1G, H) without acid phosphatase activity (data not shown). To assess vesicular compartments, muscle sections were labelled with antibodies raised against late endosomes/lysosomes (LAMP-1), early endosomes (rab5), recycling endosomes (TfR) or trans-Golgi network (Golgizone). In the lysosomal storage disorder of AMD, most fibres showed intense immunoreactivity for LAMP-1 (Fig. 1F) indicative of lysosomal proliferation. The intensity of LAMP-1 staining corresponded to the degree of vacuolation and glycogen storage. This is shown on serial sections stained for H 1 E (Fig. 1D), PAS (Fig. 1E) and LAMP-1 (Fig. 1F). In addition, LAMP-1 immunoreactivity was intense at the sarcolemma of most fibres (Figs. 1F, 2D). The pattern of Golgi-zone (green in Fig. 2B), TfR (Fig. 1I) and rab5 (Fig. 1K) immunoreactivity was similar to LAMP-1. However, sarcolemmal TfR (Fig. 1I), rab5 (Fig. 1K) and Golgi-zone (Fig. 2B) staining was less intense than LAMP-1. Thus, this staining pattern would suggest proliferation of all vesicular compartments examined in AMD. In contrast, there was no evidence of proliferation of any of these compartments in normal muscle, phosphorylase or PFK deficiency. In normal muscle, faint dot-like and patchy sarcolemmal Golgi-zone staining was present (green and yellow in co-localisation with GLUT4 in Fig. 2A), and this was similar to phosphorylase (Fig. 2C) and PFK deficiency (data not shown). Similar to Golgi-zone, weak dotlike LAMP-1 and TfR immunoreactivity was present while there was no detectable rab5 staining (data not shown). To assess the distribution of GLUT4 and its relationship to vesicles of the trans-Golgi network and the endosomal/ lysosomal system, muscle sections labelled with antibodies against GLUT4 and either Golgi-zone, LAMP-1, rab5 or TfR were evaluated using confocal microscopy. In normal muscle, GLUT4 staining was present in small dots and short segments of sarcolemma (red, yellow in co-localisation with Golgi-zone in Fig. 2A), which was similar to fibres with normal glycogen in AMD (Fig. 2B). GLUT4 immunoreactivity was intense in vacuolated glycogen storing fibres in
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Fig. 1. Morphology and glycogen storage in muscular disorders of glucose and glycogen metabolism. In AMD, vacuoles with glycogen storage and lysosomal activity were prominent on serial sections stained with H 1 E (A,D), PAS (B,E), and acid phosphatase (C, asterisk for orientation). In phosphorylase deficiency, subsarcolemmal glycogen was present in small blebs (arrowheads) on H 1 E (G) and PAS (H) stained serial sections. In AMD, serial sections were immunostained with antibodies to proteins of the endosomal/lysosomal system. Labelling with antibodies to the lysosomal marker LAMP-1 showed intense immunoreactivity predominantly in vacuolated fibres and widespread sarcolemmal staining (F, serial section to D and E). Immunostaining for TfR (I, serial section to D–F), a marker for recycling endosomes, and rab5 (K, serial section to H 1 E stained J), an early endosome marker, revealed a pattern similar to LAMP-1 albeit with weaker sarcolemmal immunoreactivity (arrow heads in K) and prominent positive patches in the centre of fibres (arrow in K) that were basophilic on H 1 E stained serial sections (arrow in J). Abbreviations: H 1 E: haematoxylin and eosin; PAS: periodic-acid Schiff; AP: acid phosphatase; LAMP-1: lysosomal associated membrane protein-1; TfR: transferrin receptor.
AMD (red in Fig. 2B, D), and staining was slightly more intense at the sarcolemma in phosphorylase (red in Fig. 2C) and PFK deficiency (data not shown). To determine GLUT4 and Golgi-zone distribution, sections were stained with GLUT4 and Golgi-zone antibodies. In AMD, GLUT4 and Golgi-zone staining partially co-localised in patches within glycogen storing fibres (yellow in Fig. 2B). This was in contrast to normal muscle (yellow in Fig. 2A) and normal looking fibres in AMD (yellow in Fig. 2B) where dot-like
co-localisation was observed, and it also differed from phosphorylase and PFK deficiency, which showed only partial dot-like co-localisation (yellow in Fig. 2C). Dual labelling with GLUT4 and LAMP-1 to assess GLUT4 in relation to late endosomes and lysosomes revealed partial co-staining in AMD (yellow in Fig. 2D), and this pattern was similar to GLUT4 and Golgi-zone dual labelling. There was no colocalisation in normal muscle, phosphorylase or PFK deficiency (data not shown). Assessing GLUT4 and early endo-
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Fig. 2. Distribution of GLUT4 in relation to vesicle populations. Muscle sections dual labelled with antibodies against GLUT4 and markers of the trans-Golgi network (Golgi) or the endosomal/lysosomal system were assessed using confocal microscopy. In normal muscle (A), GLUT4 (red) and Golgi (green) colocalised extensively (yellow on merged images) in dots and partially in patches of sarcolemma (arrows). In AMD (B, serial section to Fig. 1 A–C, asterisk for orientation), normal looking fibres showed a similar staining pattern (arrow head) while cytoplasmic GLUT4 expression (red) was intense and partially colocalised with Golgi (green) in vacuolated fibres (yellow on merged images). In phosphorylase deficiency (C, serial section to Fig. 1G,H) sarcolemmal GLUT4 staining (red) was slightly more intense than in normal muscle and co-localisation with Golgi (green) was partial. There was no GLUT4 immunoreactivity around subsarcolemmal glycogen accumulation (arrows in C, compare to PAS stained serial section Fig. 1H). AMD muscle sections dual labelled with antibodies to GLUT4 (red) and markers for late endosomes and lysosomes (LAMP-1, green in D), early endosomes (rab5, green in E) or recycling endosomes (TfR, green in F) revealed partial co-localisation (yellow) on merged images. There was no sarcolemmal co-localisation with any of the markers. GLUT4 and TfR or rab5 co-localised predominantly in patches in the centre of fibres (arrows in E and F) whereas in the periphery of fibres there was only dot-like GLUT4 staining. There was dot-like co-localisation of GLUT4 and TfR in normal looking fibres in AMD (arrow heads in F).
somes or recycling endosomes, GLUT4 and rab5 (yellow in Fig. 2E) or TfR staining (yellow in Fig. 2F) did show colocalisation in patches albeit in only approximately 10% of vacuolated fibres in AMD. These patches were localised within the fibres whereas dot-like GLUT4 staining not colocalising with either rab5 (red in Fig. 2E) or TfR (red in Fig. 2F) was present in the periphery of these fibres. In addition, in AMD, dot-like co-localisation of GLUT4 and TfR was noted in normal looking fibres (yellow in Fig. 2F). There was no detectable co-staining of GLUT4 with rab5 or TfR in normal muscle, phosphorylase or PFK deficiency (data not shown).
4. Discussion In this study the intracellular distribution of GLUT4 and of vesicles of the trans-Golgi network and the endosomal/ lysosomal system was examined in three different disorders
of glucose metabolism, and normal muscle. Our results indicate that in normal adult skeletal muscle, GLUT4 is present in a vesicular compartment expressing membrane proteins of the trans-Golgi network. This is in accord with data from cultured myoblasts [7], adipocytes [6,14–16] and transfected CHO cells [17]. Our data do not confirm an association of GLUT4 with recycling endosomes as has been reported for adipocytes [8,18]. Cultured cells may differ from adult skeletal muscle. However, staining of muscle with the antibodies used in our study was weak (TfR and LAMP-1) or not detectable (rab5). Thus, our immunohistochemical method may not be sensitive enough to detect these proteins when the vesicular systems are not proliferated. In skeletal muscle of patients with phosphorylase or PFK deficiency glycogen accumulates free in the cytoplasm as a consequence of impaired glycogenolysis, or glycolytic glucose metabolism, respectively [10]. Staining for endosomal/lysosomal markers and the trans-Golgi network was not
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different to normal muscle suggesting there was no vesicular proliferation. The distribution of GLUT4 in either disorder was only marginally different compared to normal muscle with slightly more intense sarcolemmal staining. Glycogen provides substrate for anaerobic glycolysis and subsequent aerobic mitochondrial ATP generation with dynamic exercise of sufficient intensity. In phosphorylase deficiency, the block of glycogen breakdown has been shown to impair substrate supply to the mitochondrial respiratory chain, which was partially corrected by glucose infusions [19]. This suggests that GLUT4 up-regulation in muscle relies on insulin secretion in response to systemic rather than intracellular glucose levels. Thus, increased sarcolemmal GLUT4 may have been the consequence of increased insulin release in an attempt to improve glucose supplies. In PFK deficiency, the physiological oscillations of insulin secretion in response to glucose were not observed [20]. Whether this is associated with an overall decrease in glucose transport is not clear. Muscle biopsies in our study were obtained at metabolic rest. While exercise is known to lead to up-regulation of GLUT4 in normal muscle [21] and in tetraplegic patients [22], there are to our knowledge no studies examining the influence of exercise on GLUT4 expression in phosphorylase or PFK deficiency. Furthermore, glycogen inhibition of GLUT4 mediated glucose transport may also play an as yet unknown role in these disorders [23]. It would be interesting to correlate GLUT4 expression, glycogen content and insulin secretion in these disorders. In contrast to phosphorylase and PFK deficiency, in AMD glycogen is stored largely in proliferated lysosomes which is reflected in intense LAMP-1 immunoreactivity and acid phosphatase activity. Our results suggest that AMD does not only cause lysosomal proliferation but also affects endosomes and the trans-Golgi network, vesicle populations linked to lysosomes. It is not clear whether acid maltase is directly involved in the function of these vesicles or whether these changes are secondary to lysosomal pathology. All endosomal/lysosomal markers were present at the sarcolemma raising the possibility that these vesicles may fuse with the plasma membrane, e.g. with receptor-ligand uptake and recycling of receptors [5]. Interestingly, lysosomal fusion with the plasma membrane was recognized as being potentially involved in wound healing [24], and release of lysosomal acid hydrolases was shown in activated platelets [25]. Our results could indicate that lysosomal fusion with the sarcolemma also takes place in muscle possibly releasing lysosomal contents including glycogen. In AMD, intense GLUT4 immunoreactivity was noted in glycogen storing fibres. This is surprising, since a negative feedback of glycogen on GLUT4 expression has been described [23]. However, in GLUT4 over-expressing transgenic mice muscular glycogen content was found to be greater compared to wild-type littermates and was more readily metabolised during exercise [26]. GLUT4 immunoreactivity was partially found to correlate with the prolifer-
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ated vesicular compartments suggesting they all contain GLUT4. This may be secondary to lysosomal dysfunction caused by AMD. However, as endosomes and lysosomes are likely part of a dynamic vesicular system it would be tempting to speculate that the function of acid maltase may not be limited to lysosomes. The precise role of acid maltase in skeletal muscle is not completely understood. It has been shown by in vitro studies that acid maltase is capable to fully digest glycogen [27,28], and in acid maltase knock-out mice and cultured cells from AMD patients restoration of adequate enzyme levels normalised glycogen load [29,30]. This implies an important role for acid maltase in the metabolism of glycogen. However, in fibroblasts the contribution of acid maltase towards glycogenolysis appeared to be minimal [11]. It has also been suggested that acid maltase is important for the regulation of lysosomal autophagy [11]. Recently, a defect in LAMP-2, a lysosomal membrane protein related to LAMP-1, was reported to underlie Xlinked vacuolar cardiomyopathy and myopathy, or Danon’s disease [31], and LAMP-2 deficient mice developed a similar phenotype [32]. While in these patients acid maltase activity is normal, this structural defect in an important lysosomal membrane protein can give rise to a phenotype (vacuoles filled with glycogen and autophagic material) very similar to that of AMD. Conversely, altered trafficking and metabolism of LAMP-1 was found in fibroblasts from patients with AMD which links acid maltase to this structural lysosomal membrane protein [33]. The mechanisms by which acid maltase could be regulating lysosomal function remain unclear. A possible link between lysosomal membrane function and acid maltase activity remains to be investigated. Acknowledgements The authors are grateful to K. Kappes-Horn for her support. References [1] Musi N, Fujii N, Hirshman MF, et al. AMP-activated protein kinase (AMPK) is activated in muscle of subjects with type 2 diabetes during exercise. Diabetes 2001;50:921–927. [2] Czech MP, Corvera S. Signaling mechanisms that regulate glucose transport. J Biol Chem 1999;274:1865–1868. [3] Pessin JE, Thurmond DC, Elmendorf JS, Coker KJ, Okada S. Molecular basis of insulin-stimulated GLUT4 vesicle trafficking. J Biol Chem 1999;274:2593–2596. [4] Charron MJ, Katz EB, Olson AL. GLUT4 gene regulation and manipulation. J Biol Chem 1999;274:3253–3256. [5] Mellman I. Endocytosis and molecular sorting. Ann Rev Cell Dev Biol 1996;12:575–625. [6] Slot JW, Geuze HJ, Gigengack S, Lienhard GE, James DE. Immunolocalization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol 1991;113:123–135. [7] Ralston E, Ploug T. GLUT4 in cultured skeletal muscle tubes is segregated from the transferrin receptor and stored in vesicles associated with the TGN. J Cell Sci 1996;109:2967–2978.
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[8] Livingstone C, James DE, Rice JE, Hanpeter D, Gould GW. Compartment ablation analysis of the insulin responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes. Biochem J 1996;315:487–495. [9] Slot JW, Geuze HJ, Gigengack S, James DE, Lienhard GE. Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat. Proc Natl Acad Sci USA 1991;88:7815–7829. [10] DiMauro S, Tsujino S. Nonlysosomal glycogenoses. In: Engel AG, Franzini-Armstrong C, editors. Myology: basic and clinical, 2nd ed.. New York, NY: McGraw-Hill, 1994. pp. 1557–1561. [11] Engel AG, Hirschhorn R. Acid maltase deficiency. In: Engel AG, Franzini-Armstrong C, editors. Myology: basic and clinical, 2nd ed.. New York, NY: McGraw-Hill, 1994. pp. 1533–1553. [12] Engel AG. The muscle biopsy. In: Engel AG, Franzini-Armstrong C, editors. Myology: basic and clinical, 2nd ed.. New York, NY: McGraw-Hill, 1994. pp. 822–883. [13] Dubowitz V. Muscle biopsy: a practical approach. 2nd ed.. London: Baillere Tindall, 1985. [14] Chinni SR, Shisheva A. Arrest of endosomes acidification by bafilomycin A1 mimics insulin action on GLUT4 translocation in 3T3-L1 adipocytes. Biochem J 1999;339:599–606. [15] Smith RM, Charron MJ, Shah N, Lodish HF, Jarett L. Immunoelectron microscopic demonstration of insulin-stimulated translocation of glucose transporters to the plasma membrane of isolated rat adipocytes and masking of the carboxyl-terminal epitope of intracellular GLUT4. Proc Natl Acad Sci USA 1991;88:6893–6897. [16] Herman GA, Bonzelius F, Cieutat AM, Kelly RB. A distinct class of intracellular storage vesicles, identified by expression of the glucose transporter GLUT4. Proc Natl Acad Sci USA 1994;91:12750–12754. [17] Wei ML, Bonzelius F, Scully RM, Kelly RB, Herman GA. GLUT4 and transferrin receptor are differentially sorted along the endocytic pathway in CHO cells. J Cell Biol 1998;140:565–575. [18] Martin S, Tellam J, Livingstone C, Slot JW, Gould GW, James DE. The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin-sensitive cells. J Cell Biol 1996;134:625–635. [19] Haller RG, Lewis SF, Cook JD, Blomqvist CG. Phosphorylase deficiency impairs muscle oxidative metabolism. Ann Neurol 1985;17:196–199. [20] Ristow M, Carlqvist H, Hebinck J, et al. Deficiency of muscle phosphofructo-1-kinase/muscle subtype in humans is associated with impairment of insulin secretory oscillations. Diabetes 1999;48:1557– 1561. [21] Ren JM, Semenkovich CF, Gulve EA, Gao J, Holloszy JO. Exercise induces rapid increases in GLUT4 expression, glucose transport capa-
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Effect of acid maltase deficiency on the endosomal/lysosomal system and glucose transporter 4 M. Orth a,b,*, R.R. Mundegar c a
b
University Department of Clinical Neuroscience, Royal Free and University College Medical School, London, UK University Department of Clinical and Experimental Epilepsy, Institute of Neurology, University College London, Box 19, Queen Square, London WC1N 3BG, UK c Institut fu¨r Physiologie II, University of Bonn, Wilhelmstr. 31, D-53111 Bonn, Germany Received 18 March 2002; received in revised form 4 July 2002; accepted 17 July 2002
Abstract Membrane bound glycogen storage in muscle is characteristic for the lysosomal storage disorder acid maltase (acid a-glucosidase) deficiency while in phosphofructokinase and phosphorylase deficiency, glycogen is stored free in the cytoplasm. Using immunohistochemistry, we examined whether acid maltase deficiency had an effect on early endosomes, recycling endosomes and trans-Golgi network, vesicle systems linked to lysosomes. Vacuolated glycogen containing fibres stained intensely for the lysosomal marker lysosomal-membrane-protein-1 within fibres and at the sarcolemma. There was a similar increase in immunoreactivity for markers of early endosomes (rab5), recycling endosomes (transferrin receptor) and the trans-Golgi network. In acid maltase deficiency, but not in normal muscle or other glycogenoses, staining for the insulin responsive glucose transporter 4 was markedly increased and partially co-localised with all vesicular markers. Our results suggest an effect of acid maltase deficiency extending to various vesicle systems linked to lysosomes. The enzyme defect may also affect the homoeostasis of receptors cycling through these organelles such as glucose transporter 4. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Vacuolar myopathy; GLUT4; Acid maltase; Glycogen storage; Endosome; Lysosome; Trans-Golgi network
1. Introduction Phosphorylase deficiency, phosphofructokinase (PFK) deficiency and acid maltase (acid a-glucosidase) deficiency (AMD) are associated with glycogen accumulation in skeletal muscle. Phosphorylase and PFK play well-defined roles in glycogenolysis and glycolysis, and their deficiency affects energy production pathways. Glycogen is stored non-membrane bound within the cytoplasm of phosphorylase or PFK deficient muscle. AMD is a lysosomal storage disorder. The role of acid maltase in skeletal muscle is not completely understood. However, the enzyme is thought to be able to degrade glycogen during its metabolic turnover within lysosomes. Hence, in AMD glycogen accumulates within lysosomes. In muscle and adipose cells, glucose and glycogen metabolism are linked to glucose transporter 4 (GLUT4). Glucose uptake increases both with exercise independent of insulin [1] and when insulin triggers the translocation of GLUT4
* Corresponding author. Tel.: 144-207-8373611, ext. 4272; fax: 144207-278-8772. E-mail address: [email protected] (M. Orth).
from cytoplasmic vesicle pools to the plasma membrane [2–4]. Glucose can then bind to GLUT4 and the receptorligand complex is taken up into the cell by endocytosis. There is evidence to suggest that the receptor separates from its substrate in early endosomes and is passed on to recycling endosomes and, subsequently, stored in the GLUT4-containing vesicular compartment [5]. In adipocytes and myoblasts, this compartment has been shown to express proteins characteristic for the trans-Golgi network [6,7], endosomes, and recycling endosomes [8,9]. Intracellular glycogen contents possibly down-regulate GLUT4 expression and thus contribute to GLUT4 homoeostasis [23]. To our knowledge, there are no reports on the expression of GLUT4 in skeletal muscle of patients with glucose and glycogen utilisation disorders. In these patients, an altered GLUT4 expression could help circumvent metabolic blocks and maintain energy requirements or reflect abnormalities in the endosomal/lysosomal pathway. We compared by immunohistochemistry GLUT4 localisation in normal adult skeletal muscle with that from patients with deficiencies of phosphorylase, PFK or AMD, respectively. We correlated expression of GLUT4 with proteins typically found in the membranes of early endosomes
0960-8966/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0960-896 6(02)00186-4
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(rab5), recycling endosomes (transferrin receptor, TfR), late endosomes and lysosomes (lysosomal associated membrane protein-1), and a marker for the trans-Golgi network. 2. Material and methods 2.1. Muscle biopsies Muscle biopsies were examined from five patients with adult-onset AMD, two patients with phosphorylase deficiency, one patient with PFK deficiency, and two normal controls. Diagnosis was based on clinical examination, electrophysiology and muscle biopsies applying routine histological and histochemical techniques [12,13]. The diagnoses of phosphorylase, PFK and AMD deficiency were confirmed biochemically using standard methods. 2.2. Antibodies and immunohistochemistry The following primary antibodies were used: anti-lysosomal associated membrane protein-1 (PharMingen, San Diego, USA, dilution 1/200), anti-TfR (Chemicon, Temecula, USA, 1/5), anti-Golgi zone (Chemicon, 1/200), antirab5 (Transduction Laboratories, Lexington, USA, 1/20), anti-GLUT4 (Chemicon, 1/2000). With the exception of anti-GLUT4, which is a rabbit polyclonal antibody, all antibodies are mouse monoclonal. Seven micrometer transverse sections of muscle biopsies, snap-frozen in liquid nitrogen cooled isopentane, were collected on cover slips coated with 3-aminopropyltriethoxysilan and fixed in acetone for 1 min at 2208C. Blocking of unspecific binding sites with 10% normal horse serum in phosphate-buffered saline (PBS) for 20 min was followed by incubation with the primary antibodies at room temperature for 24 h in a humid chamber. After three washes in PBS, mouse monoclonal antibodies were incubated with biotinylated horse anti-mouse secondary antibodies (Vector Laboratories, Burlingame, USA). Polyclonal rabbit antibodies were developed with biotinylated horse anti-rabbit secondary antibodies (Vector Laboratories). After another three washes in PBS, the sections were incubated with an avidin–biotin complex followed by developing for 5 min with 0.5 mg/ml 3,3 0 diaminobenzidine in PBS and 1 ml/ml H2O2 using the Vectastain-Kit (Vector Laboratories). For fluorescence staining, sections were blocked with 10% normal goat serum. Antibodies raised in mouse were developed for 1 h with Alexa 488 goat anti-mouse IgG conjugate (Molecular Probes, Eugene, USA, 1/200), while the rabbit antibody was detected with Alexa 568 goat antirabbit IgG conjugate (Molecular Probes, 1/1000). Sections were analysed and photographed using a Zeiss Fluorescence Axioplan microscope and a Zeiss MC 80 photocamera. For confocal microscopy, slides were evaluated with a Krypton–Argon laser (BioRad MRC 600) attached to an Olympus BH2-RFCA fluorescence microscope.
Specificity controls consisted of substitution of the primary antibody by irrelevant monoclonal antibodies of the same isotype and species and in the same concentration as the monoclonal primary antibodies, and of replacement of polyclonal antibodies by non-immune serum.
3. Results Muscle sections of patients with AMD, phosphorylase or PFK deficiency, and normal controls, were stained with haematoxylin/eosin (HE) to determine morphology, periodic-acid Schiff (PAS) to assess glycogen content and acid phosphatase to examine lysosomal activity. In AMD, 40–60% of muscle fibres were vacuolated (Fig. 1A, D), showed increased glycogen storage (Fig. 1B, E) and marked acid phosphatase activity (Fig. 1C). In phosphorylase or PFK deficiency, glycogen accumulated in small subsarcolemmal blebs (Fig. 1G, H) without acid phosphatase activity (data not shown). To assess vesicular compartments, muscle sections were labelled with antibodies raised against late endosomes/lysosomes (LAMP-1), early endosomes (rab5), recycling endosomes (TfR) or trans-Golgi network (Golgizone). In the lysosomal storage disorder of AMD, most fibres showed intense immunoreactivity for LAMP-1 (Fig. 1F) indicative of lysosomal proliferation. The intensity of LAMP-1 staining corresponded to the degree of vacuolation and glycogen storage. This is shown on serial sections stained for H 1 E (Fig. 1D), PAS (Fig. 1E) and LAMP-1 (Fig. 1F). In addition, LAMP-1 immunoreactivity was intense at the sarcolemma of most fibres (Figs. 1F, 2D). The pattern of Golgi-zone (green in Fig. 2B), TfR (Fig. 1I) and rab5 (Fig. 1K) immunoreactivity was similar to LAMP-1. However, sarcolemmal TfR (Fig. 1I), rab5 (Fig. 1K) and Golgi-zone (Fig. 2B) staining was less intense than LAMP-1. Thus, this staining pattern would suggest proliferation of all vesicular compartments examined in AMD. In contrast, there was no evidence of proliferation of any of these compartments in normal muscle, phosphorylase or PFK deficiency. In normal muscle, faint dot-like and patchy sarcolemmal Golgi-zone staining was present (green and yellow in co-localisation with GLUT4 in Fig. 2A), and this was similar to phosphorylase (Fig. 2C) and PFK deficiency (data not shown). Similar to Golgi-zone, weak dotlike LAMP-1 and TfR immunoreactivity was present while there was no detectable rab5 staining (data not shown). To assess the distribution of GLUT4 and its relationship to vesicles of the trans-Golgi network and the endosomal/ lysosomal system, muscle sections labelled with antibodies against GLUT4 and either Golgi-zone, LAMP-1, rab5 or TfR were evaluated using confocal microscopy. In normal muscle, GLUT4 staining was present in small dots and short segments of sarcolemma (red, yellow in co-localisation with Golgi-zone in Fig. 2A), which was similar to fibres with normal glycogen in AMD (Fig. 2B). GLUT4 immunoreactivity was intense in vacuolated glycogen storing fibres in
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Fig. 1. Morphology and glycogen storage in muscular disorders of glucose and glycogen metabolism. In AMD, vacuoles with glycogen storage and lysosomal activity were prominent on serial sections stained with H 1 E (A,D), PAS (B,E), and acid phosphatase (C, asterisk for orientation). In phosphorylase deficiency, subsarcolemmal glycogen was present in small blebs (arrowheads) on H 1 E (G) and PAS (H) stained serial sections. In AMD, serial sections were immunostained with antibodies to proteins of the endosomal/lysosomal system. Labelling with antibodies to the lysosomal marker LAMP-1 showed intense immunoreactivity predominantly in vacuolated fibres and widespread sarcolemmal staining (F, serial section to D and E). Immunostaining for TfR (I, serial section to D–F), a marker for recycling endosomes, and rab5 (K, serial section to H 1 E stained J), an early endosome marker, revealed a pattern similar to LAMP-1 albeit with weaker sarcolemmal immunoreactivity (arrow heads in K) and prominent positive patches in the centre of fibres (arrow in K) that were basophilic on H 1 E stained serial sections (arrow in J). Abbreviations: H 1 E: haematoxylin and eosin; PAS: periodic-acid Schiff; AP: acid phosphatase; LAMP-1: lysosomal associated membrane protein-1; TfR: transferrin receptor.
AMD (red in Fig. 2B, D), and staining was slightly more intense at the sarcolemma in phosphorylase (red in Fig. 2C) and PFK deficiency (data not shown). To determine GLUT4 and Golgi-zone distribution, sections were stained with GLUT4 and Golgi-zone antibodies. In AMD, GLUT4 and Golgi-zone staining partially co-localised in patches within glycogen storing fibres (yellow in Fig. 2B). This was in contrast to normal muscle (yellow in Fig. 2A) and normal looking fibres in AMD (yellow in Fig. 2B) where dot-like
co-localisation was observed, and it also differed from phosphorylase and PFK deficiency, which showed only partial dot-like co-localisation (yellow in Fig. 2C). Dual labelling with GLUT4 and LAMP-1 to assess GLUT4 in relation to late endosomes and lysosomes revealed partial co-staining in AMD (yellow in Fig. 2D), and this pattern was similar to GLUT4 and Golgi-zone dual labelling. There was no colocalisation in normal muscle, phosphorylase or PFK deficiency (data not shown). Assessing GLUT4 and early endo-
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Fig. 2. Distribution of GLUT4 in relation to vesicle populations. Muscle sections dual labelled with antibodies against GLUT4 and markers of the trans-Golgi network (Golgi) or the endosomal/lysosomal system were assessed using confocal microscopy. In normal muscle (A), GLUT4 (red) and Golgi (green) colocalised extensively (yellow on merged images) in dots and partially in patches of sarcolemma (arrows). In AMD (B, serial section to Fig. 1 A–C, asterisk for orientation), normal looking fibres showed a similar staining pattern (arrow head) while cytoplasmic GLUT4 expression (red) was intense and partially colocalised with Golgi (green) in vacuolated fibres (yellow on merged images). In phosphorylase deficiency (C, serial section to Fig. 1G,H) sarcolemmal GLUT4 staining (red) was slightly more intense than in normal muscle and co-localisation with Golgi (green) was partial. There was no GLUT4 immunoreactivity around subsarcolemmal glycogen accumulation (arrows in C, compare to PAS stained serial section Fig. 1H). AMD muscle sections dual labelled with antibodies to GLUT4 (red) and markers for late endosomes and lysosomes (LAMP-1, green in D), early endosomes (rab5, green in E) or recycling endosomes (TfR, green in F) revealed partial co-localisation (yellow) on merged images. There was no sarcolemmal co-localisation with any of the markers. GLUT4 and TfR or rab5 co-localised predominantly in patches in the centre of fibres (arrows in E and F) whereas in the periphery of fibres there was only dot-like GLUT4 staining. There was dot-like co-localisation of GLUT4 and TfR in normal looking fibres in AMD (arrow heads in F).
somes or recycling endosomes, GLUT4 and rab5 (yellow in Fig. 2E) or TfR staining (yellow in Fig. 2F) did show colocalisation in patches albeit in only approximately 10% of vacuolated fibres in AMD. These patches were localised within the fibres whereas dot-like GLUT4 staining not colocalising with either rab5 (red in Fig. 2E) or TfR (red in Fig. 2F) was present in the periphery of these fibres. In addition, in AMD, dot-like co-localisation of GLUT4 and TfR was noted in normal looking fibres (yellow in Fig. 2F). There was no detectable co-staining of GLUT4 with rab5 or TfR in normal muscle, phosphorylase or PFK deficiency (data not shown).
4. Discussion In this study the intracellular distribution of GLUT4 and of vesicles of the trans-Golgi network and the endosomal/ lysosomal system was examined in three different disorders
of glucose metabolism, and normal muscle. Our results indicate that in normal adult skeletal muscle, GLUT4 is present in a vesicular compartment expressing membrane proteins of the trans-Golgi network. This is in accord with data from cultured myoblasts [7], adipocytes [6,14–16] and transfected CHO cells [17]. Our data do not confirm an association of GLUT4 with recycling endosomes as has been reported for adipocytes [8,18]. Cultured cells may differ from adult skeletal muscle. However, staining of muscle with the antibodies used in our study was weak (TfR and LAMP-1) or not detectable (rab5). Thus, our immunohistochemical method may not be sensitive enough to detect these proteins when the vesicular systems are not proliferated. In skeletal muscle of patients with phosphorylase or PFK deficiency glycogen accumulates free in the cytoplasm as a consequence of impaired glycogenolysis, or glycolytic glucose metabolism, respectively [10]. Staining for endosomal/lysosomal markers and the trans-Golgi network was not
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different to normal muscle suggesting there was no vesicular proliferation. The distribution of GLUT4 in either disorder was only marginally different compared to normal muscle with slightly more intense sarcolemmal staining. Glycogen provides substrate for anaerobic glycolysis and subsequent aerobic mitochondrial ATP generation with dynamic exercise of sufficient intensity. In phosphorylase deficiency, the block of glycogen breakdown has been shown to impair substrate supply to the mitochondrial respiratory chain, which was partially corrected by glucose infusions [19]. This suggests that GLUT4 up-regulation in muscle relies on insulin secretion in response to systemic rather than intracellular glucose levels. Thus, increased sarcolemmal GLUT4 may have been the consequence of increased insulin release in an attempt to improve glucose supplies. In PFK deficiency, the physiological oscillations of insulin secretion in response to glucose were not observed [20]. Whether this is associated with an overall decrease in glucose transport is not clear. Muscle biopsies in our study were obtained at metabolic rest. While exercise is known to lead to up-regulation of GLUT4 in normal muscle [21] and in tetraplegic patients [22], there are to our knowledge no studies examining the influence of exercise on GLUT4 expression in phosphorylase or PFK deficiency. Furthermore, glycogen inhibition of GLUT4 mediated glucose transport may also play an as yet unknown role in these disorders [23]. It would be interesting to correlate GLUT4 expression, glycogen content and insulin secretion in these disorders. In contrast to phosphorylase and PFK deficiency, in AMD glycogen is stored largely in proliferated lysosomes which is reflected in intense LAMP-1 immunoreactivity and acid phosphatase activity. Our results suggest that AMD does not only cause lysosomal proliferation but also affects endosomes and the trans-Golgi network, vesicle populations linked to lysosomes. It is not clear whether acid maltase is directly involved in the function of these vesicles or whether these changes are secondary to lysosomal pathology. All endosomal/lysosomal markers were present at the sarcolemma raising the possibility that these vesicles may fuse with the plasma membrane, e.g. with receptor-ligand uptake and recycling of receptors [5]. Interestingly, lysosomal fusion with the plasma membrane was recognized as being potentially involved in wound healing [24], and release of lysosomal acid hydrolases was shown in activated platelets [25]. Our results could indicate that lysosomal fusion with the sarcolemma also takes place in muscle possibly releasing lysosomal contents including glycogen. In AMD, intense GLUT4 immunoreactivity was noted in glycogen storing fibres. This is surprising, since a negative feedback of glycogen on GLUT4 expression has been described [23]. However, in GLUT4 over-expressing transgenic mice muscular glycogen content was found to be greater compared to wild-type littermates and was more readily metabolised during exercise [26]. GLUT4 immunoreactivity was partially found to correlate with the prolifer-
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ated vesicular compartments suggesting they all contain GLUT4. This may be secondary to lysosomal dysfunction caused by AMD. However, as endosomes and lysosomes are likely part of a dynamic vesicular system it would be tempting to speculate that the function of acid maltase may not be limited to lysosomes. The precise role of acid maltase in skeletal muscle is not completely understood. It has been shown by in vitro studies that acid maltase is capable to fully digest glycogen [27,28], and in acid maltase knock-out mice and cultured cells from AMD patients restoration of adequate enzyme levels normalised glycogen load [29,30]. This implies an important role for acid maltase in the metabolism of glycogen. However, in fibroblasts the contribution of acid maltase towards glycogenolysis appeared to be minimal [11]. It has also been suggested that acid maltase is important for the regulation of lysosomal autophagy [11]. Recently, a defect in LAMP-2, a lysosomal membrane protein related to LAMP-1, was reported to underlie Xlinked vacuolar cardiomyopathy and myopathy, or Danon’s disease [31], and LAMP-2 deficient mice developed a similar phenotype [32]. While in these patients acid maltase activity is normal, this structural defect in an important lysosomal membrane protein can give rise to a phenotype (vacuoles filled with glycogen and autophagic material) very similar to that of AMD. Conversely, altered trafficking and metabolism of LAMP-1 was found in fibroblasts from patients with AMD which links acid maltase to this structural lysosomal membrane protein [33]. The mechanisms by which acid maltase could be regulating lysosomal function remain unclear. A possible link between lysosomal membrane function and acid maltase activity remains to be investigated. Acknowledgements The authors are grateful to K. Kappes-Horn for her support. References [1] Musi N, Fujii N, Hirshman MF, et al. AMP-activated protein kinase (AMPK) is activated in muscle of subjects with type 2 diabetes during exercise. Diabetes 2001;50:921–927. [2] Czech MP, Corvera S. Signaling mechanisms that regulate glucose transport. J Biol Chem 1999;274:1865–1868. [3] Pessin JE, Thurmond DC, Elmendorf JS, Coker KJ, Okada S. Molecular basis of insulin-stimulated GLUT4 vesicle trafficking. J Biol Chem 1999;274:2593–2596. [4] Charron MJ, Katz EB, Olson AL. GLUT4 gene regulation and manipulation. J Biol Chem 1999;274:3253–3256. [5] Mellman I. Endocytosis and molecular sorting. Ann Rev Cell Dev Biol 1996;12:575–625. [6] Slot JW, Geuze HJ, Gigengack S, Lienhard GE, James DE. Immunolocalization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol 1991;113:123–135. [7] Ralston E, Ploug T. GLUT4 in cultured skeletal muscle tubes is segregated from the transferrin receptor and stored in vesicles associated with the TGN. J Cell Sci 1996;109:2967–2978.
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