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Expression of the GLUT1 and GLUT9 facilitative glucose transporters in embryonic chondroblasts and mature chondrocytes in ovine articular cartilage
Cell Biology International 29 (2005) 249e260 www.elsevier.com/locate/cellbi
Expression of the GLUT1 and GLUT9 facilitative glucose transporters in embryonic chondroblasts and mature chondrocytes in ovine articular cartilage A. Mobasheria,*, H. Dobsona, S.L. Masona, F. Cullinghama, M. Shakibaeib,c, J.F. Moleyd, K.H. Moleyd a
Connective Tissue, Molecular Pathogenesis, Reproduction and Stress Research Groups, Department of Veterinary Preclinical Sciences, Faculty of Veterinary Science, University of Liverpool, Liverpool L69 7ZJ, UK b Institute of Anatomy, Ludwig-Maximilians-University Munich, 80336 Munich, Germany c Institute of Anatomy, Charite´ Medicine University Berlin, Campus Benjamin Franklin, D-14195 Berlin, Germany d Departments of Surgery, Obstetrics, Gynecology, Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA Received 13 July 2004; revised 15 November 2004; accepted 23 November 2004
Abstract Glucose transport across the chondrocyte membrane is essential for chondrogenesis and the development of the skeletal system. We have previously used RTePCR to show that fully developed human articular chondrocytes express transcripts for the GLUT1 and GLUT9 glucose transporters. In this study we report on the expression and immunohistochemical localization of the GLUT1 and GLUT9 proteins in embryonic and mature ovine cartilage. We also provide Western blot evidence for GLUT1 and GLUT9 expression in mature ovine chondrocytes. Ovine embryos (developmental stages E32 to E36 and E42 to E45) were obtained from pregnant ewes humanely killed by injection with sodium pentobarbitone. Embryos were fixed and processed for immunohistochemistry. Polyclonal antibodies to GLUT1 and GLUT9 revealed that both transporters are expressed in developing chondrocytes in ovine embryos and in the superficial, middle and deep layers of ovine cartilage from mature animals. GLUT1 expression was observed in erythrocytes and organs including heart, liver, and kidney. GLUT9 was also found in heart, kidney and liver. Western blotting confirmed the presence of the GLUT1 protein which migrated between the 50 and 64 kDa markers and two specific GLUT9 bands migrating under the 50 and 60 kDa markers, respectively. The presence of GLUT1 and GLUT9 in developing joints of ovine embryos suggests that these proteins may be important in glucose delivery to developing chondroblasts. Expression of these GLUT isoforms may be an important bioenergetic adaptation for chondrocytes in the extracellular matrix of developing cartilage. Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Ovidae; Cartilage; Chondroblast; Chondrocyte; Embryonic development; GLUT1; GLUT9; Immunohistochemistry
1. Introduction Glucose is an essential source of energy for chondroblasts during chondrogenesis, embryonic growth and * Corresponding author. Tel.: C44 151 794 4284; fax: C44 151 794 4243. E-mail address: [email protected] (A. Mobasheri).
foetal development (Macheda et al., 2002; Mobasheri et al., 2002b) and is vital for articular and growth plate chondrocytes during post-natal skeletal development (Ohara et al., 2001; Wang et al., 1999). Glucose also plays a key role in cartilage extracellular matrix synthesis as a precursor for glycosaminoglycans (Mobasheri et al., 2002b). The facilitated transport of glucose across the chondrocyte membrane represents the rate-limiting step
1065-6995/$ - see front matter Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2004.11.024
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in glucose metabolism and is essential for the functional integrity of articulating joints (Mobasheri et al., 2002b; Shikhman et al., 2001). Mammalian cells transport glucose through the GLUT/SLC2A and SGLT/SLC5A families of glucose transporters (Wood and Trayhurn, 2003). The tissue distribution of glucose transporters is not constant throughout development (Santalucia et al., 1992). High levels of GLUT1 and GLUT3 are present in a wide range of foetal tissues, with expression of these transporters greatly decreased after birth in many cell types. Abundant levels of the GLUT1 and GLUT3 proteins are also present in pre-implantation mouse embryos, since glucose is the main substrate consumed (Pantaleon and Kaye, 1998; Pantaleon et al., 2001). During the early period of organ formation (i.e. brain, heart, skeletal muscle and kidney) GLUT1 is responsible for glucose supply to the dividing and differentiating cells (Matsumoto et al., 1995; Santalucia et al., 1992). There is evidence that GLUT8 (the novel insulin-like growth factor I (IGF-I) and insulin regulated glucose transporter) and GLUT9 both play a role in pre-implantation development (Carayannopoulos et al., 2000, 2004; Wyman et al., 2003). Recent studies suggest that GLUT1 and GLUT9 are present in chondrocytes derived from fully developed human articular cartilage (Mobasheri et al., 2002a; Richardson et al., 2003; Shikhman et al., 2001). However, virtually nothing is known about the expression of these two glucose transporter isoforms during cartilage development. It has not been determined if GLUT1 and GLUT9 are present in articular cartilage and musculoskeletal tissues of post-implantation embryos. Thus the hypothesis we wish to test in this study is that glucose transporters are important for chondrogenesis during embryonic development and that GLUT1 and GLUT9 may be important in this process. Accordingly, in this paper, we report on the developmental expression of GLUT1 and GLUT9 in embryonic, juvenile and mature ovine cartilage and provide evidence for the presence of these glucose transporter proteins in embryonic ovine chondroblasts and mature ovine articular chondrocytes.
cross, a cross between Lleyns and Bleu de Maine to give a Blue cross ewe. The former animals reach up to 75 kg in weight but the latter are heavier (reaching 85e90 kg in weight). Both are breeds capable of bringing over two lambs per ewe on average. All ewes were fed the same food and kept under identical conditions. The sheep were fed grass all summer and hay (moist hay; 70% dry matter) all winter. The abattoir sheep (same two breeds) were fed a commercially acceptable diet. 2.3. Outline of study design and source of ovine tissues
Unless otherwise stated, all chemicals and reagents used in this study were molecular biology grade and were purchased from Sigma Biosciences (Poole, UK). Secondary antibodies and immunohistochemical kit reagents were purchased from DakoCytomation (Ely, UK) and Vector Laboratories (Peterborough, UK).
The principal objective of this study was to examine the expression and distribution of the GLUT1 and GLUT9 glucose transporters in embryonic and mature cartilage by immunohistochemistry. The experimental design included animals in early developmental stages (E32 to E36; E42 to E45) and later when skeletal growth accelerates or is complete. A total of six foetuses were removed following humane euthanasia of pregnant ewes by injection with 0.75 ml/ kg body weight of Euthatal solution (Rhoˆne Me´rieux, Harlow, UK) containing 200 mg/ml pentobarbitone sodium BP (VET). All procedures were carried out strictly following ethical local guidelines. The foetal dates described were assigned by comparison of foetal dimensions to wellestablished and published values for prenatal sheep embryos (Green, 1946). The embryos were from a range of developmental stages (E32 to E36 and E42 to E45). The smallest foetuses (developmental age between E32 and E36, nZ2) were immediately fixed in neutral buffered formalin. The age of the remaining foetuses (nZ4) ranged from approximately E42 to E45. Fulldepth cartilage plugs were excised from the forelimbs of mature animals (metacarpal joints of pregnant ewes or abattoir material) using a cork borer. The abattoir material was derived from more than twelve mixed male and female joints but the data shown are restricted to three joints. All tissues intended for immunohistochemical studies, including whole E32eE36 embryos, hind limbs of E42eE45 embryos and cartilage derived from mature animals (abattoir material from three animals ranging from 2 to 3 years in age) were fixed for 48 h in neutral buffered formalin and decalcified in 10% EDTA for up to 3 weeks at room temperature (RT). Paraffin embedding was carried out in-house by our histopathology service. Tissues were cut (6 mm thickness) and mounted on microscope glass slides coated with 3aminopropyl-triethoxysilane (APES) (VWR International, Poole, UK).
2.2. Animal breeds, care and nutrition
2.4. Antibodies to GLUT1 and GLUT9
Two types of ewes were used in this study: Lleyns ewes which are a lowland breed of sheep and Lleyn
Polyclonal antibodies against GLUT1 were donated by Dr S.A. Baldwin (Department of Biochemistry and
2. Materials and methods 2.1. Chemicals
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Molecular Biology, University of Leeds, UK). The GLUT1 antiserum was developed in rabbits against the C-terminus of rat-GLUT1 (residues 477e492) and affinity-purified. We have confirmed that the GLUT1 antibody is highly suitable for immunohistochemistry as it recognizes GLUT1 across a diverse number of mammalian species including human, canine, rat, mouse, marmot, bovine, ovine, equine and elephant. There are numerous publications in the literature that report successful application of this polyclonal antibody in studies of human and animal tissues (Brown et al., 1988; Davies et al., 1987, 1990; Froehner et al., 1988; Kasanicki et al., 1987; Takakura et al., 1991; Yi et al., 1992). Rabbit polyclonal antibodies against human GLUT9 (Augustin et al., 2004; Carayannopoulos et al., 2004) were developed in the laboratories of Dr J.F. Moley and Dr K.H. Moley (Washington University School of Medicine, St. Louis, USA). Although the GLUT9 antisera are less well characterized, they have recently been affinity purified and successfully used to study GLUT9 expression (Augustin et al., 2004).
2.5. Immunohistochemistry Two immunohistochemical methods were adopted to accommodate different tissue adhesive characteristics at the different developmental stages studied. Slides were heated at 60 C for 15 min to improve tissue adhesion to the glass slides which were then dewaxed in xylene for 20 min before the tissue sections were rehydrated in distilled water via a graded series of ethanol baths (1 min in each of the following ethanol solutions: 100%, 95%, 70%, 50%). Heat-assisted antigen retrieval was carried out in a 10 mM citrate buffer (pH 6.0) (Shi et al., 1991). Sections were either heated at full power in a 750 W microwave oven for three rounds of 12 min or in a 95 C water bath for a total period of 35 min. Most of the experimental data presented was obtained using peroxidase-conjugated secondary antibody (a component of the DakoCytomation EnVisionCDual Link System, Peroxidase (DABC) Kit; manufacturer’s code K4065). In immunohistochemical experiments on mature articular cartilage alkaline phosphatase conjugated secondary antibody was used (a component of DAKO EnVisionÔ Double stain Kit, K 1395). Endogenous peroxidase activity was blocked for 15 min at RT using DAKO endogenous peroxidase block. Endogenous alkaline phosphatase was quenched by treatment with 1.25 mM levamisole solution (Vector Laboratories). The slides were incubated for 1 h at RT with 20% normal goat serum (NGS) in phosphate-buffered saline (PBS) containing 1% bovine serum albumin to block nonspecific antibody binding. Slides were incubated at 4 C overnight with primary polyclonal antibodies to human GLUT1 and human GLUT9. Both antibodies were
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diluted 1:200 in PBS. After three 10-min washes in PBSe Tween (0.05%), the sections were incubated with secondary horseradish peroxidase-labelled polymer or alkaline phosphatase-labelled conjugated to affinitypurified goat anti-rabbit immunoglobulins (DAKO) for 30 min at RT. The sections exposed to horseradish peroxidase-labelled secondary antibody were washed three times for 10 min in PBSeTween before applying liquid DABC Chromogen (DAKO; 3,3#-diaminobenzidine solution) for up to 3 min. The development of the brown-coloured reaction was stopped by rinsing in PBS containing 0.05% sodium azide. The sections exposed to alkaline phosphatase-labelled secondary antibody were washed three times for 10 min in PBS and alkaline phosphatase active sites were revealed using Fast-Red TR/Naphthol AS-MX as precipitating agent. Sections were then immersed for 5 min in a bath of aqueous haematoxylin (DAKO Code No. S 3309) to counter stain cell nuclei. Fast-Red slides were mounted in aqueous medium (H. D. Supplies, U.K.). DABC Chromogen slides were mounted in 1,3-diethyl-8-phenylxanthine (DPX) (VWR International). In every experiment, sections were incubated with several controls to rule out the possibility of non-specific binding and to demonstrate the specificity of each antibody used. Appropriate control experiments were performed by incubating slides with non-immune rabbit serum (Sigma), omission of primary antibody or inclusion of irrelevant antiserum (DAKO). Digital images were captured using a Nikon Microphot-FX microscope fitted with a Nikon DXM1200 digital camera and saved as uncompressed 1280!1024 pixel TIFF files. Images were analysed by Scion Image (version 4.0.2) based on NIH Image for Macintosh. The immunohistochemical data were compiled using CorelDraw (version 10.427) and exported as PDF or JPEG files.
2.6. Ovine chondrocyte isolation and culture Primary cultures of ovine articular chondrocytes were prepared from articular cartilage derived from metacarpal joints of pregnant ewes or abattoir material. Briefly, the cartilage was excised, rinsed with Hanks’ solution and incubated overnight with 0.2% (v/v) type II collagenase in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1000 mg Lÿ1 glucose, 10% foetal calf serum and 1% antibiotic/antimycotic solution. The resulting cell suspension was separated from non-dissociated tissue fragments by filtration through a nylon mesh with a pore diameter of 70 mm. Aliquots of this cell suspension (viability 95% or higher as determined by Trypan blue dye exclusion assay) were transferred to 75-cm2 flasks and the cells were passaged only once to preserve the chondrocyte phenotype. Culture medium was changed every 3 days.
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2.7. Preparation of whole cell lysates and Western blot analysis of GLUT1 and GLUT9 in ovine chondrocytes Freshly isolated and passage 1 ovine chondrocytes were homogenized in lysis buffer (50 mM Tris/HCl, pH 7.2, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 0.01% (v/v) aprotinin, 4 mg/ml pepstatin A, 10 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, PMSF) on ice for 60 min. Nuclei were removed by centrifugation and the protein content of the resulting supernatant (whole-cell lysate) was determined using the Bio-Rad Detergent Compatible (DC) kit and bovine serum albumin as standard. Equal amounts of total protein were separated on 12% polyacrylamide gels under reducing conditions, transferred onto nitrocellulose, blocked with 5% (w/v) non-fat dry milk in phosphate-buffered saline/0.1% Tween 20 (PBS-Tween) and probed overnight at 4 C with antibodies to GLUT1 and GLUT9 (both diluted 1 in 1000 in PBSeTween). After washing in PBSeTween blots were incubated with peroxidase-labelled polymer conjugated to goat antirabbit immunoglobulins in PBSeTween buffer and developed by chemiluminescence using the SuperSignal Western kit (Pierce).
3. Results To determine the expression and distribution of GLUT1 and GLUT9 in developing ovine articular cartilage, we performed immunohistochemistry on embryos ranging between E32eE36 and E42eE45 using polyclonal antibodies to GLUT1 and GLUT9. We also examined articular cartilage from joints of 2e3-year-old sheep for expression of GLUT1 and GLUT9 using immunohistochemistry and Western blot analysis. Selected micrographs from the immunohistochemical experiments described are summarized in Figs. 1e3. Microwave oven assisted antigen retrieval in 10 mM citrate buffer (pH 6.0) (Shi et al., 1992) was essential prior to the immunohistochemical procedure. This approach exposed the antigens which significantly improved the immunoreactivity of GLUT1 and GLUT9 in formalin fixed ovine tissue sections. 3.1. Immunohistochemical localization of GLUT1 and GLUT9 in tissues of E32eE36 ovine embryos GLUT1 and GLUT9 were both found to be present in E32eE36 ovine embryos (Fig. 1). GLUT1 was abundantly expressed in many tissues including most regions of the brain and gut (data not shown) the kidneys, heart and liver (Fig. 1G,J,M, respectively). GLUT1 expression
was detected in condensing mesenchymal cells and chondroblasts in the spinal column, ribs and in mineralizing bone in the skull and jawbone (Fig. 1A,D). GLUT1 expression was particularly prominent in erythrocytes in blood vessels of the developing heart, liver and kidney. GLUT9 was detected in condensing mesenchymal cells in the spine and chondroblasts and bone cells in the mineralizing jawbone (Fig. 1B,E). The expression of GLUT9 in developing cardiac muscle and liver (Fig. 1K,M) was high and comparable with the expression of GLUT1. GLUT9 immunostaining in kidneys of E32eE36 ovine embryos was weak (Fig. 1H) and restricted to structures resembling to proximal tubules of developing nephrons. Non-specific immunostaining did not appear to occur when the primary GLUT1 and GLUT9 antibodies were omitted from the immunohistochemical protocol (Fig. 1; control panels C,F,I,L,O) or when slides were incubated with nonimmune serum (data not shown). 3.2. Immunohistochemical localization of GLUT1 and GLUT9 in connective tissues of E42 to E45 ovine embryos To determine whether GLUT1 and GLUT9 are present in developing ovine articular cartilage from E42eE45 embryos, we performed immunohistochemistry as described above. However, due to the increased size and complexity of the embryos at this developmental stage, we focused on connective tissues of hind-limbs. GLUT1 and GLUT9 were present in chondroblasts in femoral head, metacarpal and epiphyseal cartilage (Fig. 2A,B,D,E,G,H). High levels of GLUT1 and GLUT9 were found in the bone marrow of the femur (Fig. 2J,K) and in skeletal muscle (Fig. 2M,N). Slides used as negative controls demonstrated the absence of nonspecific immunostaining when the slides were incubated with non-immune rabbit serum (Fig. 2C,F,I,L,O) or when the primary polyclonal antibody was omitted from the immunohistochemical protocol (data not shown). Higher magnification images of GLUT1 and GLUT9 expression in chondrocytes from femoral head cartilage and epiphyseal cartilage from limbs of E42eE45 embryos are shown in Fig. 3. 3.3. Immunohistochemical localization of GLUT1 and GLUT9 in mature ovine articular cartilage GLUT1 and GLUT9 were both detected in the superficial, middle and deep layers of mature ovine articular cartilage (Fig. 4). Positive GLUT1 and GLUT9 expression was recorded in more than six independent experiments on mature cartilage specimens (from a total of three mature animals - the sex of the adult animals was not known as the tissues were derived from an
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Fig. 1. Immunoperoxidase localization of GLUT1 and GLUT9 in selected tissues of E32eE36 ovine embryos. The images shown are representative sections taken from two ovine foetuses at this developmental stage. Expression of GLUT1 and GLUT9 is shown in the cartilage and mineralizing bone of the lower mandible (panels A, B) and in the spine (panels D, E). High levels of GLUT1 and GLUT9 expression were also detected in the heart and liver (panels J, K, M, N). GLUT1 immunostaining in the developing kidney was observed in basolateral membranes of renal tubules and erythrocytes (panel G). GLUT9 immunostaining in the developing kidney was weak and restricted to nephron structures resembling proximal tubules (panel H). The negative controls demonstrate that non-specific immunostaining did not occur when the primary antibody was omitted from the immunohistochemical protocol. Bars represent 100 mm and nuclei were counterstained with haematoxylin.
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Fig. 2. Immunoperoxidase localization of GLUT1 and GLUT9 in connective tissues of E42eE45 ovine embryos. The images shown are representative sections taken from four ovine foetuses at this developmental stage. Expression of GLUT1 and GLUT9 is shown in femoral head, metacarpal and epiphyseal cartilage and mineralizing bone of the femur (panels A, B, D, E, G, H, J, K). Expression of GLUT1 and GLUT9 is also evident in skeletal muscle. The negative controls demonstrate that non-specific immunostaining did not occur when the slides were incubated with non-immune rabbit serum (panels C, F, I, L, O). Nuclei were counterstained with haematoxylin. Bars represent 100 mm.
abattoir). GLUT1 and GLUT9 were both strongly expressed in the superficial and middle zones of articular cartilage, a distribution pattern that resembled the immunostaining observed in the femoral and metacarpal cartilage of E42eE45 embryos. Closer examination
revealed that GLUT1 and GLUT9 were present not only in the plasma membrane but also inside cells in an undetermined location. In addition to chondroblasts of ovine embryos, GLUT1 and GLUT9 immunoreactivity was also observed in keratinocytes of skin and the
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Fig. 3. High-magnification immunohistochemical micrographs showing the cellular localization of GLUT1 and GLUT9 in articular cartilage and epiphyseal cartilage from joints of E42eE45 embryos. Plasma membrane and cytoplasmic GLUT1 staining is evident in panels A and C. Cytoplasmic and nuclear membrane localization is seen with anti-GLUT9 antibodies (panels B and D).
developing hoof (data not shown). The results of GLUT1 and GLUT9 immunohistochemical experiments on the three selected developmental stages are summarized in Table 1. 3.4. Western blot analysis of GLUT1 and GLUT9 protein expression in ovine chondrocytes To demonstrate endogenous expression of GLUT1 and GLUT9 proteins in ovine articular chondrocytes, we performed Western blot analysis of whole-cell lysates from freshly isolated, and passage 1 cultured cells. GLUT1: A specific and highly immunoreactive GLUT1 Table 1 Summary of the developmental expression of GLUT1 and GLUT9 in articular cartilage. Developmental stage
GLUT1
GLUT9
Embryonic cartilage (E32eE36) Embryonic cartilage (E42eE45) Mature cartilage
CCCC CCCC CC
CCC CCC CC
The data presented show the relative amount of expression of the individual GLUT isoforms. Expression:CCCC, abundant;CCC, strong;CC, moderate;C, low.
protein band was detected in ovine chondrocytes migrating broadly between the 50 and 64 kDa markers (Fig. 5A). GLUT9: Two immunoreactive bands were detected with the GLUT9 antiserum. A prominent band was seen at 60 kDa (presumably the highly glycosylated form of GLUT9) and a less abundant lower molecular weight band migrating under 50 kDa (a less glycosylated form of the protein or a different splice variant) (Fig. 5B).
4. Discussion The major findings of this investigation indicate that in addition to GLUT3, and GLUT12, developing and mature mammalian chondrocytes also express GLUT1 and the recently described GLUT9 glucose transporter. However, unlike GLUT12, GLUT9 and GLUT1, appear to be expressed during development and in maturity in articular cartilage and may be important for the maintenance and homeostasis of fully developed cartilage. Sugar transport across the plasma membrane of mammalian cells is mediated by members of the GLUT/SLC2A family of facilitative sugar transporters
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Fig. 4. Immunohistochemical localization of GLUT1 and GLUT9 in mature ovine articular cartilage. The images shown are representative sections taken from three abattoir animals ranging from 2 to 3 years in age. Fast-Red TR/Naphthol AS-MX was used as the precipitating agent in these experiments to reveal sites of alkaline phosphatase activity and hence GLUT1 and GLUT9 expression. GLUT1 and GLUT9 positive cells were detected in the middle and deep zones with very low levels in calcified cartilage and endothelial cells lining capillaries running through the deep zone. Nuclei were counterstained with haematoxylin.
and the SGLT/SLC5A and family of NaC-dependent sugar transporters (Wood and Trayhurn, 2003). Fourteen members of the GLUT/SLC2A family have been cloned in humans (Wood and Trayhurn, 2003; Wu and Freeze, 2002) and it is becoming apparent that the expression of GLUT isoforms is developmentally regulated in many tissues and organs. GLUT proteins play key roles in glucose homeostasis in livestock species (Hocquette and Abe, 2000). In vivo studies have demonstrated that some GLUTs (especially
GLUT4) are regulated by nutritional and hormonal factors in pigs, cattle, lactating cows and goats and throughout foetal life in the placenta and tissues of lambs and calves (Hocquette and Abe, 2000; Hocquette et al., 1996a). In addition there are important differences in GLUT4 expression between oxidative and glycolytic muscles in livestock animals (Hocquette et al., 1995, 1996b). Therefore, these observations suggest that any changes in GLUT expression and activity influence nutrient partitioning and tissue metabolism in
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Fig. 5. Expression of GLUT1 and GLUT9 proteins in ovine chondrocytes. Whole-cell lysates were prepared as described in Section 2, separated by SDSePAGE, and blotted onto nitrocellulose membranes. Rabbit anti-GLUT1 and GLUT9 antibodies were used to probe the blots by chemiluminescence.
food-producing animals and may affect the quality and quantity of milk and meat (Hocquette and Abe, 2000). Optimal growth and development of musculoskeletal structures in livestock species is important for skeletal stability and meat yield and quality (Geay et al., 2001). GLUT proteins are also likely to influence the development of the musculoskeletal structures of loadbearing synovial joints including muscle, articular cartilage, tendon and ligament. Thus far, there have been no published studies on the expression of any of the well studied GLUTs (GLUT1e5) or recently identified GLUTs (GLUT6e14) in cartilage during embryonic development; only one recent study has shown that GLUT1 and GLUT12 proteins are expressed in foetal rat chondrocytes (Macheda et al., 2002). Almost nothing is known about the developmental expression of glucose transporters during embryogenesis in food-producing animals. This is an important area of research that may have been hindered due to the technical difficulties associated with working with large non-rodent embryos. The study of facilitative glucose transporters in connective tissues requires experience of handling calcified tissues in addition to high quality probes and antibodies for in situ hybridization and immunological experiments to identify the GLUT isoforms expressed at the mRNA and protein levels (Hocquette and Abe, 2000). The importance of glucose as the ubiquitous energy currency within developing organisms is undeniably
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linked to its abundance in nature. In the early organogenesis period high affinity glucose transporters may be required because embryonic mammalian cells may have to rely upon anaerobic glycolysis (Matsumoto et al., 1995). It is well known that the transcription factor HIF-1 alpha (HIF-1a) is essential for chondrocyte growth arrest and survival in vivo (Schipani et al., 2001) and for the maintenance of anaerobic glycolysis and matrix synthesis in epiphyseal chondrocytes (Pfander et al., 2003). HIF-1a is necessary for regulating glycolysis under anaerobic conditions since HIF-1a-null chondrocytes are unable to maintain ATP levels in hypoxic microenvironments (Schipani et al., 2001), presumably due to their inability to activate HIF-1a target genes including GLUT1, GLUT3 (Badr et al., 1999; Behrooz and Ismail-Beigi, 1997; Chen et al., 2001), glycolytic enzymes and angiogenic factors such as vascular endothelial growth factor (VEGF) (Semenza, 1999, 2000). Glucose is also particularly important for anabolic activities of other mesenchymal cells that differentiate into specialized cells of the musculoskeletal system (Vannucci et al., 2000). Provision of glucose as an essential metabolite and structural precursor to growing tissues is particularly important during foetal development, when cells are rapidly dividing and differentiating (Matsumoto et al., 1995; Pantaleon and Kaye, 1998; Santalucia et al., 1992; Vannucci, 1994). Previous studies have shown that the GLUT1 isoform is present at high levels in almost all foetal tissues; with levels decreasing after birth and disappearing in most fully developed tissues except the liver, erythrocytes and the blood-brain barrier (Vannucci and Vannucci, 2000). Recent immunohistochemical studies in chondrocytes of 3-day-old and 7-day-old rats have revealed intense immunoreactivity of GLUT1 in growth plate chondrocytes (Ohara et al., 2001). Immunohistochemistry has also been used to show that the GLUT12 protein, a recently identified member of the sugar transporter family, and GLUT1, the best studied member of the GLUT/SLC2A family are both expressed in articular cartilage during rat foetal development (Macheda et al., 2002). The expression of GLUT12 in insulin-responsive tissues supports a potential role for GLUT12 in the provision of glucose to these tissues before the appearance of GLUT4 which has been shown to be expressed in the mouse growth plate and in the developing mouse embryo (Vannucci et al., 2000; Wang et al., 1999). Evidence also suggests that IGF-I exerts ‘‘insulin-like’’ anabolic actions on hypertrophic growth plate chondrocytes during longitudinal bone growth (Wang et al., 1999). Glucose is also important in fully developed articular cartilage due to the poor vascularization and highly glycolytic nature of the tissue, a situation that is further exacerbated by low oxygen tensions and ongoing anaerobic glycolysis by chondrocytes (Mobasheri
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et al., 2002b; Otte, 1991; Rajpurohit et al., 2002). Therefore, even modest changes in glucose concentrations in the extracellular microenvironment of chondrocytes could impair anabolic and catabolic activities (Mobasheri et al., 2002b; Shikhman et al., 2001). Fully developed adult chondrocytes express multiple isoforms of the GLUT/SLC2A family of glucose transporters including GLUT1, GLUT3, GLUT5, GLUT6, GLUT8, GLUT9, GLUT10, GLUT11 and GLUT12 (Mobasheri et al., 2002a,b; Richardson et al., 2003; Shikhman et al., 2001). The reason for such GLUT isoform diversity in chondrocytes has not yet been satisfactorily explained. However, several hypotheses have been proposed: the presence of GLUT1 in chondrocytes has been linked to the acute requirement of these cells for glycolytic energy metabolism under the low oxygen tension conditions that are prevalent in avascular load-bearing articular cartilage (Mobasheri et al., 2002b). GLUT1 has also been shown to be a cytokine inducible glucose transporter in cartilage since it may be induced by catabolic, pro-inflammatory cytokines (Phillips et al., 2005; Richardson et al., 2003; Shikhman et al., 2001, 2004). GLUT9 is a novel member of the glucose transporter family which was recently identified by screening internet-based Expressed Sequence Tag (EST) and high throughput genome sequence databases for novel sequences homologous to previously known glucose transporters (Phay et al., 2000b). Northern blot analysis has revealed that the GLUT9 gene produces, by alternative splicing, three mRNA species which are abundantly detected in kidney and liver, but are also present at low levels in several other tissues including placenta, lung, blood leukocytes, heart, and skeletal muscle (Phay et al., 2000a) (updated information may be found on the NCBI AceView site: http://www.ncbi. nlm.nih.gov/IEB/Research/Acembly/av.cgi?dbZ33&cZ Gene&lZSLC2A9). The results presented in this paper confirm our previous immunohistochemical observations in human cartilage in which GLUT9 has been reported to be expressed (Mobasheri et al., 2002a; Richardson et al., 2003). Therefore, expression of multiple GLUT isoforms (including GLUT9) may be an important physiological and bioenergetic adaptation for chondrocytes in the extracellular matrix in developing and mature cartilage (Mobasheri et al., 2002a). GLUT9 is most abundantly expressed in liver and kidney (Phay et al., 2000a,b), both highly metabolic tissues capable of gluconeogenesis. The precise physiological role of GLUT9 in cartilage is unclear but ongoing studies suggest that it may be involved in glucose uptake, extracellular matrix protein glycosylation or glycogenesis. Some intracellular localization was observed with both GLUT1 and GLUT9 antibodies in this study. However, we did not attempt to determine the exact location(s) of the intracellular staining. Chondrocytes
possess an extensive endoplasmic reticulum and Golgi complex which are actively engaged in glycosylation of newly synthesized matrix glycosaminoglycans. Intracellular GLUT1 has been observed in inner-cell-mass plasma membranes of pre-implantation mouse embryos and associated with metabolism in oocytes (Pantaleon et al., 2001). It is therefore plausible that some of the intracellular GLUT1 and GLUT9 protein detected are functional intracellular or ‘‘organellar’’ glucose transporters. It is also possible that this intracellular staining represents newly synthesized proteins en route to the plasma membrane or subcellular storage vesicles. The presence of GLUT1 and GLUT9 in embryonic ovine chondroblasts supports a critical role for these glucose transporters and possibly others (GLUT3 and GLUT12) in ovine cartilage development. The GLUT isoforms implicated in embryonic chondroblasts may be involved in transporting hexose and pentose sugars (i.e. glucose and fructose, respectively), sulphated sugars and possibly also dehydroascorbic acid (vitamin C) which is involved in hydroxyproline and hydroxylysine synthesis for incorporation into newly synthesized cartilage matrix collagens. These glucose transporters are probably important for the maintenance and homeostasis of fully developed cartilage matrix. Chondrocytes also possess the capacity for gluconeogenesis via the glyoxylate pathway and some of the GLUTs may be involved in the glucose fluxes that may occur in physiological scenarios where these pathways are functional.
Acknowledgements This work was financially supported by grants from the University of Liverpool Research Development Fund, The Pet Plan Charitable Trust (UK) (A.M.), the National Institutes of Health (J.F.M., K.H.M.) and the Deutsche Forschungsgemeinschaft (M.S., Grants Sh 48/ 2-4, Sh 48/2-5).
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Expression of the GLUT1 and GLUT9 facilitative glucose transporters in embryonic chondroblasts and mature chondrocytes in ovine articular cartilage A. Mobasheria,*, H. Dobsona, S.L. Masona, F. Cullinghama, M. Shakibaeib,c, J.F. Moleyd, K.H. Moleyd a
Connective Tissue, Molecular Pathogenesis, Reproduction and Stress Research Groups, Department of Veterinary Preclinical Sciences, Faculty of Veterinary Science, University of Liverpool, Liverpool L69 7ZJ, UK b Institute of Anatomy, Ludwig-Maximilians-University Munich, 80336 Munich, Germany c Institute of Anatomy, Charite´ Medicine University Berlin, Campus Benjamin Franklin, D-14195 Berlin, Germany d Departments of Surgery, Obstetrics, Gynecology, Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA Received 13 July 2004; revised 15 November 2004; accepted 23 November 2004
Abstract Glucose transport across the chondrocyte membrane is essential for chondrogenesis and the development of the skeletal system. We have previously used RTePCR to show that fully developed human articular chondrocytes express transcripts for the GLUT1 and GLUT9 glucose transporters. In this study we report on the expression and immunohistochemical localization of the GLUT1 and GLUT9 proteins in embryonic and mature ovine cartilage. We also provide Western blot evidence for GLUT1 and GLUT9 expression in mature ovine chondrocytes. Ovine embryos (developmental stages E32 to E36 and E42 to E45) were obtained from pregnant ewes humanely killed by injection with sodium pentobarbitone. Embryos were fixed and processed for immunohistochemistry. Polyclonal antibodies to GLUT1 and GLUT9 revealed that both transporters are expressed in developing chondrocytes in ovine embryos and in the superficial, middle and deep layers of ovine cartilage from mature animals. GLUT1 expression was observed in erythrocytes and organs including heart, liver, and kidney. GLUT9 was also found in heart, kidney and liver. Western blotting confirmed the presence of the GLUT1 protein which migrated between the 50 and 64 kDa markers and two specific GLUT9 bands migrating under the 50 and 60 kDa markers, respectively. The presence of GLUT1 and GLUT9 in developing joints of ovine embryos suggests that these proteins may be important in glucose delivery to developing chondroblasts. Expression of these GLUT isoforms may be an important bioenergetic adaptation for chondrocytes in the extracellular matrix of developing cartilage. Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Ovidae; Cartilage; Chondroblast; Chondrocyte; Embryonic development; GLUT1; GLUT9; Immunohistochemistry
1. Introduction Glucose is an essential source of energy for chondroblasts during chondrogenesis, embryonic growth and * Corresponding author. Tel.: C44 151 794 4284; fax: C44 151 794 4243. E-mail address: [email protected] (A. Mobasheri).
foetal development (Macheda et al., 2002; Mobasheri et al., 2002b) and is vital for articular and growth plate chondrocytes during post-natal skeletal development (Ohara et al., 2001; Wang et al., 1999). Glucose also plays a key role in cartilage extracellular matrix synthesis as a precursor for glycosaminoglycans (Mobasheri et al., 2002b). The facilitated transport of glucose across the chondrocyte membrane represents the rate-limiting step
1065-6995/$ - see front matter Ó 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2004.11.024
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in glucose metabolism and is essential for the functional integrity of articulating joints (Mobasheri et al., 2002b; Shikhman et al., 2001). Mammalian cells transport glucose through the GLUT/SLC2A and SGLT/SLC5A families of glucose transporters (Wood and Trayhurn, 2003). The tissue distribution of glucose transporters is not constant throughout development (Santalucia et al., 1992). High levels of GLUT1 and GLUT3 are present in a wide range of foetal tissues, with expression of these transporters greatly decreased after birth in many cell types. Abundant levels of the GLUT1 and GLUT3 proteins are also present in pre-implantation mouse embryos, since glucose is the main substrate consumed (Pantaleon and Kaye, 1998; Pantaleon et al., 2001). During the early period of organ formation (i.e. brain, heart, skeletal muscle and kidney) GLUT1 is responsible for glucose supply to the dividing and differentiating cells (Matsumoto et al., 1995; Santalucia et al., 1992). There is evidence that GLUT8 (the novel insulin-like growth factor I (IGF-I) and insulin regulated glucose transporter) and GLUT9 both play a role in pre-implantation development (Carayannopoulos et al., 2000, 2004; Wyman et al., 2003). Recent studies suggest that GLUT1 and GLUT9 are present in chondrocytes derived from fully developed human articular cartilage (Mobasheri et al., 2002a; Richardson et al., 2003; Shikhman et al., 2001). However, virtually nothing is known about the expression of these two glucose transporter isoforms during cartilage development. It has not been determined if GLUT1 and GLUT9 are present in articular cartilage and musculoskeletal tissues of post-implantation embryos. Thus the hypothesis we wish to test in this study is that glucose transporters are important for chondrogenesis during embryonic development and that GLUT1 and GLUT9 may be important in this process. Accordingly, in this paper, we report on the developmental expression of GLUT1 and GLUT9 in embryonic, juvenile and mature ovine cartilage and provide evidence for the presence of these glucose transporter proteins in embryonic ovine chondroblasts and mature ovine articular chondrocytes.
cross, a cross between Lleyns and Bleu de Maine to give a Blue cross ewe. The former animals reach up to 75 kg in weight but the latter are heavier (reaching 85e90 kg in weight). Both are breeds capable of bringing over two lambs per ewe on average. All ewes were fed the same food and kept under identical conditions. The sheep were fed grass all summer and hay (moist hay; 70% dry matter) all winter. The abattoir sheep (same two breeds) were fed a commercially acceptable diet. 2.3. Outline of study design and source of ovine tissues
Unless otherwise stated, all chemicals and reagents used in this study were molecular biology grade and were purchased from Sigma Biosciences (Poole, UK). Secondary antibodies and immunohistochemical kit reagents were purchased from DakoCytomation (Ely, UK) and Vector Laboratories (Peterborough, UK).
The principal objective of this study was to examine the expression and distribution of the GLUT1 and GLUT9 glucose transporters in embryonic and mature cartilage by immunohistochemistry. The experimental design included animals in early developmental stages (E32 to E36; E42 to E45) and later when skeletal growth accelerates or is complete. A total of six foetuses were removed following humane euthanasia of pregnant ewes by injection with 0.75 ml/ kg body weight of Euthatal solution (Rhoˆne Me´rieux, Harlow, UK) containing 200 mg/ml pentobarbitone sodium BP (VET). All procedures were carried out strictly following ethical local guidelines. The foetal dates described were assigned by comparison of foetal dimensions to wellestablished and published values for prenatal sheep embryos (Green, 1946). The embryos were from a range of developmental stages (E32 to E36 and E42 to E45). The smallest foetuses (developmental age between E32 and E36, nZ2) were immediately fixed in neutral buffered formalin. The age of the remaining foetuses (nZ4) ranged from approximately E42 to E45. Fulldepth cartilage plugs were excised from the forelimbs of mature animals (metacarpal joints of pregnant ewes or abattoir material) using a cork borer. The abattoir material was derived from more than twelve mixed male and female joints but the data shown are restricted to three joints. All tissues intended for immunohistochemical studies, including whole E32eE36 embryos, hind limbs of E42eE45 embryos and cartilage derived from mature animals (abattoir material from three animals ranging from 2 to 3 years in age) were fixed for 48 h in neutral buffered formalin and decalcified in 10% EDTA for up to 3 weeks at room temperature (RT). Paraffin embedding was carried out in-house by our histopathology service. Tissues were cut (6 mm thickness) and mounted on microscope glass slides coated with 3aminopropyl-triethoxysilane (APES) (VWR International, Poole, UK).
2.2. Animal breeds, care and nutrition
2.4. Antibodies to GLUT1 and GLUT9
Two types of ewes were used in this study: Lleyns ewes which are a lowland breed of sheep and Lleyn
Polyclonal antibodies against GLUT1 were donated by Dr S.A. Baldwin (Department of Biochemistry and
2. Materials and methods 2.1. Chemicals
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Molecular Biology, University of Leeds, UK). The GLUT1 antiserum was developed in rabbits against the C-terminus of rat-GLUT1 (residues 477e492) and affinity-purified. We have confirmed that the GLUT1 antibody is highly suitable for immunohistochemistry as it recognizes GLUT1 across a diverse number of mammalian species including human, canine, rat, mouse, marmot, bovine, ovine, equine and elephant. There are numerous publications in the literature that report successful application of this polyclonal antibody in studies of human and animal tissues (Brown et al., 1988; Davies et al., 1987, 1990; Froehner et al., 1988; Kasanicki et al., 1987; Takakura et al., 1991; Yi et al., 1992). Rabbit polyclonal antibodies against human GLUT9 (Augustin et al., 2004; Carayannopoulos et al., 2004) were developed in the laboratories of Dr J.F. Moley and Dr K.H. Moley (Washington University School of Medicine, St. Louis, USA). Although the GLUT9 antisera are less well characterized, they have recently been affinity purified and successfully used to study GLUT9 expression (Augustin et al., 2004).
2.5. Immunohistochemistry Two immunohistochemical methods were adopted to accommodate different tissue adhesive characteristics at the different developmental stages studied. Slides were heated at 60 C for 15 min to improve tissue adhesion to the glass slides which were then dewaxed in xylene for 20 min before the tissue sections were rehydrated in distilled water via a graded series of ethanol baths (1 min in each of the following ethanol solutions: 100%, 95%, 70%, 50%). Heat-assisted antigen retrieval was carried out in a 10 mM citrate buffer (pH 6.0) (Shi et al., 1991). Sections were either heated at full power in a 750 W microwave oven for three rounds of 12 min or in a 95 C water bath for a total period of 35 min. Most of the experimental data presented was obtained using peroxidase-conjugated secondary antibody (a component of the DakoCytomation EnVisionCDual Link System, Peroxidase (DABC) Kit; manufacturer’s code K4065). In immunohistochemical experiments on mature articular cartilage alkaline phosphatase conjugated secondary antibody was used (a component of DAKO EnVisionÔ Double stain Kit, K 1395). Endogenous peroxidase activity was blocked for 15 min at RT using DAKO endogenous peroxidase block. Endogenous alkaline phosphatase was quenched by treatment with 1.25 mM levamisole solution (Vector Laboratories). The slides were incubated for 1 h at RT with 20% normal goat serum (NGS) in phosphate-buffered saline (PBS) containing 1% bovine serum albumin to block nonspecific antibody binding. Slides were incubated at 4 C overnight with primary polyclonal antibodies to human GLUT1 and human GLUT9. Both antibodies were
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diluted 1:200 in PBS. After three 10-min washes in PBSe Tween (0.05%), the sections were incubated with secondary horseradish peroxidase-labelled polymer or alkaline phosphatase-labelled conjugated to affinitypurified goat anti-rabbit immunoglobulins (DAKO) for 30 min at RT. The sections exposed to horseradish peroxidase-labelled secondary antibody were washed three times for 10 min in PBSeTween before applying liquid DABC Chromogen (DAKO; 3,3#-diaminobenzidine solution) for up to 3 min. The development of the brown-coloured reaction was stopped by rinsing in PBS containing 0.05% sodium azide. The sections exposed to alkaline phosphatase-labelled secondary antibody were washed three times for 10 min in PBS and alkaline phosphatase active sites were revealed using Fast-Red TR/Naphthol AS-MX as precipitating agent. Sections were then immersed for 5 min in a bath of aqueous haematoxylin (DAKO Code No. S 3309) to counter stain cell nuclei. Fast-Red slides were mounted in aqueous medium (H. D. Supplies, U.K.). DABC Chromogen slides were mounted in 1,3-diethyl-8-phenylxanthine (DPX) (VWR International). In every experiment, sections were incubated with several controls to rule out the possibility of non-specific binding and to demonstrate the specificity of each antibody used. Appropriate control experiments were performed by incubating slides with non-immune rabbit serum (Sigma), omission of primary antibody or inclusion of irrelevant antiserum (DAKO). Digital images were captured using a Nikon Microphot-FX microscope fitted with a Nikon DXM1200 digital camera and saved as uncompressed 1280!1024 pixel TIFF files. Images were analysed by Scion Image (version 4.0.2) based on NIH Image for Macintosh. The immunohistochemical data were compiled using CorelDraw (version 10.427) and exported as PDF or JPEG files.
2.6. Ovine chondrocyte isolation and culture Primary cultures of ovine articular chondrocytes were prepared from articular cartilage derived from metacarpal joints of pregnant ewes or abattoir material. Briefly, the cartilage was excised, rinsed with Hanks’ solution and incubated overnight with 0.2% (v/v) type II collagenase in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1000 mg Lÿ1 glucose, 10% foetal calf serum and 1% antibiotic/antimycotic solution. The resulting cell suspension was separated from non-dissociated tissue fragments by filtration through a nylon mesh with a pore diameter of 70 mm. Aliquots of this cell suspension (viability 95% or higher as determined by Trypan blue dye exclusion assay) were transferred to 75-cm2 flasks and the cells were passaged only once to preserve the chondrocyte phenotype. Culture medium was changed every 3 days.
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2.7. Preparation of whole cell lysates and Western blot analysis of GLUT1 and GLUT9 in ovine chondrocytes Freshly isolated and passage 1 ovine chondrocytes were homogenized in lysis buffer (50 mM Tris/HCl, pH 7.2, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 0.01% (v/v) aprotinin, 4 mg/ml pepstatin A, 10 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, PMSF) on ice for 60 min. Nuclei were removed by centrifugation and the protein content of the resulting supernatant (whole-cell lysate) was determined using the Bio-Rad Detergent Compatible (DC) kit and bovine serum albumin as standard. Equal amounts of total protein were separated on 12% polyacrylamide gels under reducing conditions, transferred onto nitrocellulose, blocked with 5% (w/v) non-fat dry milk in phosphate-buffered saline/0.1% Tween 20 (PBS-Tween) and probed overnight at 4 C with antibodies to GLUT1 and GLUT9 (both diluted 1 in 1000 in PBSeTween). After washing in PBSeTween blots were incubated with peroxidase-labelled polymer conjugated to goat antirabbit immunoglobulins in PBSeTween buffer and developed by chemiluminescence using the SuperSignal Western kit (Pierce).
3. Results To determine the expression and distribution of GLUT1 and GLUT9 in developing ovine articular cartilage, we performed immunohistochemistry on embryos ranging between E32eE36 and E42eE45 using polyclonal antibodies to GLUT1 and GLUT9. We also examined articular cartilage from joints of 2e3-year-old sheep for expression of GLUT1 and GLUT9 using immunohistochemistry and Western blot analysis. Selected micrographs from the immunohistochemical experiments described are summarized in Figs. 1e3. Microwave oven assisted antigen retrieval in 10 mM citrate buffer (pH 6.0) (Shi et al., 1992) was essential prior to the immunohistochemical procedure. This approach exposed the antigens which significantly improved the immunoreactivity of GLUT1 and GLUT9 in formalin fixed ovine tissue sections. 3.1. Immunohistochemical localization of GLUT1 and GLUT9 in tissues of E32eE36 ovine embryos GLUT1 and GLUT9 were both found to be present in E32eE36 ovine embryos (Fig. 1). GLUT1 was abundantly expressed in many tissues including most regions of the brain and gut (data not shown) the kidneys, heart and liver (Fig. 1G,J,M, respectively). GLUT1 expression
was detected in condensing mesenchymal cells and chondroblasts in the spinal column, ribs and in mineralizing bone in the skull and jawbone (Fig. 1A,D). GLUT1 expression was particularly prominent in erythrocytes in blood vessels of the developing heart, liver and kidney. GLUT9 was detected in condensing mesenchymal cells in the spine and chondroblasts and bone cells in the mineralizing jawbone (Fig. 1B,E). The expression of GLUT9 in developing cardiac muscle and liver (Fig. 1K,M) was high and comparable with the expression of GLUT1. GLUT9 immunostaining in kidneys of E32eE36 ovine embryos was weak (Fig. 1H) and restricted to structures resembling to proximal tubules of developing nephrons. Non-specific immunostaining did not appear to occur when the primary GLUT1 and GLUT9 antibodies were omitted from the immunohistochemical protocol (Fig. 1; control panels C,F,I,L,O) or when slides were incubated with nonimmune serum (data not shown). 3.2. Immunohistochemical localization of GLUT1 and GLUT9 in connective tissues of E42 to E45 ovine embryos To determine whether GLUT1 and GLUT9 are present in developing ovine articular cartilage from E42eE45 embryos, we performed immunohistochemistry as described above. However, due to the increased size and complexity of the embryos at this developmental stage, we focused on connective tissues of hind-limbs. GLUT1 and GLUT9 were present in chondroblasts in femoral head, metacarpal and epiphyseal cartilage (Fig. 2A,B,D,E,G,H). High levels of GLUT1 and GLUT9 were found in the bone marrow of the femur (Fig. 2J,K) and in skeletal muscle (Fig. 2M,N). Slides used as negative controls demonstrated the absence of nonspecific immunostaining when the slides were incubated with non-immune rabbit serum (Fig. 2C,F,I,L,O) or when the primary polyclonal antibody was omitted from the immunohistochemical protocol (data not shown). Higher magnification images of GLUT1 and GLUT9 expression in chondrocytes from femoral head cartilage and epiphyseal cartilage from limbs of E42eE45 embryos are shown in Fig. 3. 3.3. Immunohistochemical localization of GLUT1 and GLUT9 in mature ovine articular cartilage GLUT1 and GLUT9 were both detected in the superficial, middle and deep layers of mature ovine articular cartilage (Fig. 4). Positive GLUT1 and GLUT9 expression was recorded in more than six independent experiments on mature cartilage specimens (from a total of three mature animals - the sex of the adult animals was not known as the tissues were derived from an
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Fig. 1. Immunoperoxidase localization of GLUT1 and GLUT9 in selected tissues of E32eE36 ovine embryos. The images shown are representative sections taken from two ovine foetuses at this developmental stage. Expression of GLUT1 and GLUT9 is shown in the cartilage and mineralizing bone of the lower mandible (panels A, B) and in the spine (panels D, E). High levels of GLUT1 and GLUT9 expression were also detected in the heart and liver (panels J, K, M, N). GLUT1 immunostaining in the developing kidney was observed in basolateral membranes of renal tubules and erythrocytes (panel G). GLUT9 immunostaining in the developing kidney was weak and restricted to nephron structures resembling proximal tubules (panel H). The negative controls demonstrate that non-specific immunostaining did not occur when the primary antibody was omitted from the immunohistochemical protocol. Bars represent 100 mm and nuclei were counterstained with haematoxylin.
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Fig. 2. Immunoperoxidase localization of GLUT1 and GLUT9 in connective tissues of E42eE45 ovine embryos. The images shown are representative sections taken from four ovine foetuses at this developmental stage. Expression of GLUT1 and GLUT9 is shown in femoral head, metacarpal and epiphyseal cartilage and mineralizing bone of the femur (panels A, B, D, E, G, H, J, K). Expression of GLUT1 and GLUT9 is also evident in skeletal muscle. The negative controls demonstrate that non-specific immunostaining did not occur when the slides were incubated with non-immune rabbit serum (panels C, F, I, L, O). Nuclei were counterstained with haematoxylin. Bars represent 100 mm.
abattoir). GLUT1 and GLUT9 were both strongly expressed in the superficial and middle zones of articular cartilage, a distribution pattern that resembled the immunostaining observed in the femoral and metacarpal cartilage of E42eE45 embryos. Closer examination
revealed that GLUT1 and GLUT9 were present not only in the plasma membrane but also inside cells in an undetermined location. In addition to chondroblasts of ovine embryos, GLUT1 and GLUT9 immunoreactivity was also observed in keratinocytes of skin and the
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Fig. 3. High-magnification immunohistochemical micrographs showing the cellular localization of GLUT1 and GLUT9 in articular cartilage and epiphyseal cartilage from joints of E42eE45 embryos. Plasma membrane and cytoplasmic GLUT1 staining is evident in panels A and C. Cytoplasmic and nuclear membrane localization is seen with anti-GLUT9 antibodies (panels B and D).
developing hoof (data not shown). The results of GLUT1 and GLUT9 immunohistochemical experiments on the three selected developmental stages are summarized in Table 1. 3.4. Western blot analysis of GLUT1 and GLUT9 protein expression in ovine chondrocytes To demonstrate endogenous expression of GLUT1 and GLUT9 proteins in ovine articular chondrocytes, we performed Western blot analysis of whole-cell lysates from freshly isolated, and passage 1 cultured cells. GLUT1: A specific and highly immunoreactive GLUT1 Table 1 Summary of the developmental expression of GLUT1 and GLUT9 in articular cartilage. Developmental stage
GLUT1
GLUT9
Embryonic cartilage (E32eE36) Embryonic cartilage (E42eE45) Mature cartilage
CCCC CCCC CC
CCC CCC CC
The data presented show the relative amount of expression of the individual GLUT isoforms. Expression:CCCC, abundant;CCC, strong;CC, moderate;C, low.
protein band was detected in ovine chondrocytes migrating broadly between the 50 and 64 kDa markers (Fig. 5A). GLUT9: Two immunoreactive bands were detected with the GLUT9 antiserum. A prominent band was seen at 60 kDa (presumably the highly glycosylated form of GLUT9) and a less abundant lower molecular weight band migrating under 50 kDa (a less glycosylated form of the protein or a different splice variant) (Fig. 5B).
4. Discussion The major findings of this investigation indicate that in addition to GLUT3, and GLUT12, developing and mature mammalian chondrocytes also express GLUT1 and the recently described GLUT9 glucose transporter. However, unlike GLUT12, GLUT9 and GLUT1, appear to be expressed during development and in maturity in articular cartilage and may be important for the maintenance and homeostasis of fully developed cartilage. Sugar transport across the plasma membrane of mammalian cells is mediated by members of the GLUT/SLC2A family of facilitative sugar transporters
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Fig. 4. Immunohistochemical localization of GLUT1 and GLUT9 in mature ovine articular cartilage. The images shown are representative sections taken from three abattoir animals ranging from 2 to 3 years in age. Fast-Red TR/Naphthol AS-MX was used as the precipitating agent in these experiments to reveal sites of alkaline phosphatase activity and hence GLUT1 and GLUT9 expression. GLUT1 and GLUT9 positive cells were detected in the middle and deep zones with very low levels in calcified cartilage and endothelial cells lining capillaries running through the deep zone. Nuclei were counterstained with haematoxylin.
and the SGLT/SLC5A and family of NaC-dependent sugar transporters (Wood and Trayhurn, 2003). Fourteen members of the GLUT/SLC2A family have been cloned in humans (Wood and Trayhurn, 2003; Wu and Freeze, 2002) and it is becoming apparent that the expression of GLUT isoforms is developmentally regulated in many tissues and organs. GLUT proteins play key roles in glucose homeostasis in livestock species (Hocquette and Abe, 2000). In vivo studies have demonstrated that some GLUTs (especially
GLUT4) are regulated by nutritional and hormonal factors in pigs, cattle, lactating cows and goats and throughout foetal life in the placenta and tissues of lambs and calves (Hocquette and Abe, 2000; Hocquette et al., 1996a). In addition there are important differences in GLUT4 expression between oxidative and glycolytic muscles in livestock animals (Hocquette et al., 1995, 1996b). Therefore, these observations suggest that any changes in GLUT expression and activity influence nutrient partitioning and tissue metabolism in
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Fig. 5. Expression of GLUT1 and GLUT9 proteins in ovine chondrocytes. Whole-cell lysates were prepared as described in Section 2, separated by SDSePAGE, and blotted onto nitrocellulose membranes. Rabbit anti-GLUT1 and GLUT9 antibodies were used to probe the blots by chemiluminescence.
food-producing animals and may affect the quality and quantity of milk and meat (Hocquette and Abe, 2000). Optimal growth and development of musculoskeletal structures in livestock species is important for skeletal stability and meat yield and quality (Geay et al., 2001). GLUT proteins are also likely to influence the development of the musculoskeletal structures of loadbearing synovial joints including muscle, articular cartilage, tendon and ligament. Thus far, there have been no published studies on the expression of any of the well studied GLUTs (GLUT1e5) or recently identified GLUTs (GLUT6e14) in cartilage during embryonic development; only one recent study has shown that GLUT1 and GLUT12 proteins are expressed in foetal rat chondrocytes (Macheda et al., 2002). Almost nothing is known about the developmental expression of glucose transporters during embryogenesis in food-producing animals. This is an important area of research that may have been hindered due to the technical difficulties associated with working with large non-rodent embryos. The study of facilitative glucose transporters in connective tissues requires experience of handling calcified tissues in addition to high quality probes and antibodies for in situ hybridization and immunological experiments to identify the GLUT isoforms expressed at the mRNA and protein levels (Hocquette and Abe, 2000). The importance of glucose as the ubiquitous energy currency within developing organisms is undeniably
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linked to its abundance in nature. In the early organogenesis period high affinity glucose transporters may be required because embryonic mammalian cells may have to rely upon anaerobic glycolysis (Matsumoto et al., 1995). It is well known that the transcription factor HIF-1 alpha (HIF-1a) is essential for chondrocyte growth arrest and survival in vivo (Schipani et al., 2001) and for the maintenance of anaerobic glycolysis and matrix synthesis in epiphyseal chondrocytes (Pfander et al., 2003). HIF-1a is necessary for regulating glycolysis under anaerobic conditions since HIF-1a-null chondrocytes are unable to maintain ATP levels in hypoxic microenvironments (Schipani et al., 2001), presumably due to their inability to activate HIF-1a target genes including GLUT1, GLUT3 (Badr et al., 1999; Behrooz and Ismail-Beigi, 1997; Chen et al., 2001), glycolytic enzymes and angiogenic factors such as vascular endothelial growth factor (VEGF) (Semenza, 1999, 2000). Glucose is also particularly important for anabolic activities of other mesenchymal cells that differentiate into specialized cells of the musculoskeletal system (Vannucci et al., 2000). Provision of glucose as an essential metabolite and structural precursor to growing tissues is particularly important during foetal development, when cells are rapidly dividing and differentiating (Matsumoto et al., 1995; Pantaleon and Kaye, 1998; Santalucia et al., 1992; Vannucci, 1994). Previous studies have shown that the GLUT1 isoform is present at high levels in almost all foetal tissues; with levels decreasing after birth and disappearing in most fully developed tissues except the liver, erythrocytes and the blood-brain barrier (Vannucci and Vannucci, 2000). Recent immunohistochemical studies in chondrocytes of 3-day-old and 7-day-old rats have revealed intense immunoreactivity of GLUT1 in growth plate chondrocytes (Ohara et al., 2001). Immunohistochemistry has also been used to show that the GLUT12 protein, a recently identified member of the sugar transporter family, and GLUT1, the best studied member of the GLUT/SLC2A family are both expressed in articular cartilage during rat foetal development (Macheda et al., 2002). The expression of GLUT12 in insulin-responsive tissues supports a potential role for GLUT12 in the provision of glucose to these tissues before the appearance of GLUT4 which has been shown to be expressed in the mouse growth plate and in the developing mouse embryo (Vannucci et al., 2000; Wang et al., 1999). Evidence also suggests that IGF-I exerts ‘‘insulin-like’’ anabolic actions on hypertrophic growth plate chondrocytes during longitudinal bone growth (Wang et al., 1999). Glucose is also important in fully developed articular cartilage due to the poor vascularization and highly glycolytic nature of the tissue, a situation that is further exacerbated by low oxygen tensions and ongoing anaerobic glycolysis by chondrocytes (Mobasheri
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et al., 2002b; Otte, 1991; Rajpurohit et al., 2002). Therefore, even modest changes in glucose concentrations in the extracellular microenvironment of chondrocytes could impair anabolic and catabolic activities (Mobasheri et al., 2002b; Shikhman et al., 2001). Fully developed adult chondrocytes express multiple isoforms of the GLUT/SLC2A family of glucose transporters including GLUT1, GLUT3, GLUT5, GLUT6, GLUT8, GLUT9, GLUT10, GLUT11 and GLUT12 (Mobasheri et al., 2002a,b; Richardson et al., 2003; Shikhman et al., 2001). The reason for such GLUT isoform diversity in chondrocytes has not yet been satisfactorily explained. However, several hypotheses have been proposed: the presence of GLUT1 in chondrocytes has been linked to the acute requirement of these cells for glycolytic energy metabolism under the low oxygen tension conditions that are prevalent in avascular load-bearing articular cartilage (Mobasheri et al., 2002b). GLUT1 has also been shown to be a cytokine inducible glucose transporter in cartilage since it may be induced by catabolic, pro-inflammatory cytokines (Phillips et al., 2005; Richardson et al., 2003; Shikhman et al., 2001, 2004). GLUT9 is a novel member of the glucose transporter family which was recently identified by screening internet-based Expressed Sequence Tag (EST) and high throughput genome sequence databases for novel sequences homologous to previously known glucose transporters (Phay et al., 2000b). Northern blot analysis has revealed that the GLUT9 gene produces, by alternative splicing, three mRNA species which are abundantly detected in kidney and liver, but are also present at low levels in several other tissues including placenta, lung, blood leukocytes, heart, and skeletal muscle (Phay et al., 2000a) (updated information may be found on the NCBI AceView site: http://www.ncbi. nlm.nih.gov/IEB/Research/Acembly/av.cgi?dbZ33&cZ Gene&lZSLC2A9). The results presented in this paper confirm our previous immunohistochemical observations in human cartilage in which GLUT9 has been reported to be expressed (Mobasheri et al., 2002a; Richardson et al., 2003). Therefore, expression of multiple GLUT isoforms (including GLUT9) may be an important physiological and bioenergetic adaptation for chondrocytes in the extracellular matrix in developing and mature cartilage (Mobasheri et al., 2002a). GLUT9 is most abundantly expressed in liver and kidney (Phay et al., 2000a,b), both highly metabolic tissues capable of gluconeogenesis. The precise physiological role of GLUT9 in cartilage is unclear but ongoing studies suggest that it may be involved in glucose uptake, extracellular matrix protein glycosylation or glycogenesis. Some intracellular localization was observed with both GLUT1 and GLUT9 antibodies in this study. However, we did not attempt to determine the exact location(s) of the intracellular staining. Chondrocytes
possess an extensive endoplasmic reticulum and Golgi complex which are actively engaged in glycosylation of newly synthesized matrix glycosaminoglycans. Intracellular GLUT1 has been observed in inner-cell-mass plasma membranes of pre-implantation mouse embryos and associated with metabolism in oocytes (Pantaleon et al., 2001). It is therefore plausible that some of the intracellular GLUT1 and GLUT9 protein detected are functional intracellular or ‘‘organellar’’ glucose transporters. It is also possible that this intracellular staining represents newly synthesized proteins en route to the plasma membrane or subcellular storage vesicles. The presence of GLUT1 and GLUT9 in embryonic ovine chondroblasts supports a critical role for these glucose transporters and possibly others (GLUT3 and GLUT12) in ovine cartilage development. The GLUT isoforms implicated in embryonic chondroblasts may be involved in transporting hexose and pentose sugars (i.e. glucose and fructose, respectively), sulphated sugars and possibly also dehydroascorbic acid (vitamin C) which is involved in hydroxyproline and hydroxylysine synthesis for incorporation into newly synthesized cartilage matrix collagens. These glucose transporters are probably important for the maintenance and homeostasis of fully developed cartilage matrix. Chondrocytes also possess the capacity for gluconeogenesis via the glyoxylate pathway and some of the GLUTs may be involved in the glucose fluxes that may occur in physiological scenarios where these pathways are functional.
Acknowledgements This work was financially supported by grants from the University of Liverpool Research Development Fund, The Pet Plan Charitable Trust (UK) (A.M.), the National Institutes of Health (J.F.M., K.H.M.) and the Deutsche Forschungsgemeinschaft (M.S., Grants Sh 48/ 2-4, Sh 48/2-5).
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