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Differential regulation of the GLUT1 and GLUT3 glucose transporters by growth factors and pro-inflammatory cytokines in equine articular chondrocytes
The Veterinary Journal The Veterinary Journal 169 (2005) 216–222 www.elsevier.com/locate/tvjl
Differential regulation of the GLUT1 and GLUT3 glucose transporters by growth factors and pro-inflammatory cytokines in equine articular chondrocytes Toby Phillips, Ivan Ferraz 1, Susan Bell, Peter D. Clegg, Stuart D. Carter, Ali Mobasheri * Connective Tissue and Molecular Pathogenesis Research Groups, Faculty of Veterinary Science, University of Liverpool, Liverpool L69 7ZJ, UK Accepted 16 January 2004
Abstract Glucose serves as the major energy substrate for articular chondrocytes and as the main precursor for the synthesis of extracellular matrix glycosaminoglycans in cartilage. Chondrocytes have been shown to express several glucose transporter (GLUT) isoforms including GLUT1 and GLUT3. The aim of this investigation was to determine the effects of endocrine and cytokine factors on the capacity of equine articular chondrocytes for transporting 2-deoxy-D -[2, 6-3 H] glucose and on the expression levels of GLUT1 and GLUT3. Chondrocytes maintained in monolayer culture were stimulated for 24 h with TNF-a (100 ng mL1 ), IL-1b (100 ng mL1 ), IGF-I (20 ng mL1 ), TGF-b (20 ng mL1 ) and insulin (12.5 lg mL1 ) before measuring uptake of non-metabolizable 2-deoxyglucose in the presence and absence of the glucose transport inhibitor cytochalasin B. Polyclonal antibodies to GLUT1 and GLUT were used to compare GLUT1 and GLUT3 expression in stimulated and un-stimulated alginate encapsulated chondrocytes by Western blotting. Results indicated that 2-deoxyglucose uptake was inhibited by up to 95% in the presence of cytochalasin B suggesting that glucose uptake into equine chondrocytes is GLUT-mediated. Insulin had no effect on glucose uptake, but treatment with IGF-I, TGF-b, IL-1b and TNF-a resulted in a significant increase (>65%) in 2-deoxyglucose uptake compared to control values. GLUT1 was found to be increased in chondrocytes stimulated with all the growth factors and cytokines but GLUT 3 was only upregulated by IGF-I. The data presented support a critical role for glucose in the responses of equine articular chondrocytes to pro-inflammatory cytokines and anabolic endocrine factors. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Cartilage; Chondrocyte; Glucose transport; GLUT; Cytokine; Growth factor
1. Introduction Recent research suggests that osteoarticular disorders in humans and veterinary species may be directly linked to obesity and may therefore have nutritional and en-
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Corresponding author. Tel.: +44-151-794-4284; fax: +44-151-7944243. E-mail address: [email protected] (A. Mobasheri). 1 Present Address: Servicio de Reumatologia, Hospital Universitario de Canarias, Tenerife, La Cuesta, Santa Cruz de Tenerife and Departamento de Bioquımica y Biologıa Molecular, Universidad de La Laguna, Tenerife, Spain. 1090-0233/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2004.01.026
docrine abnormalities at the root of their pathogenesis. Glucose is an essential energy source for mammalian cells and in articular cartilage glucose plays a pivotal role in the physiology of the chondrocytes by driving the extracellular matrix biosynthetic machinery of this unique cell type (Wang et al., 1999; Mobasheri et al., 2002b). Glucose is also a major energy source and a precursor for the synthesis of glycosaminoglycans (Mobasheri et al., 2002a). Despite this realisation, there is limited published information about the molecular mechanisms responsible for nutrient transport across the chondrocyte membrane and their regulation by growth factors and pro-inflammatory cytokines.
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The facilitated transport of glucose and glucosederived compounds across the chondrocyte membrane represents the rate-limiting step in glucose metabolism (Shikhman et al., 2001a) and is therefore essential for chondrogenesis and the functional integrity of articulating joints. Mammalian cells transport glucose across the plasma membrane by facilitated diffusion through the glucose transporters (or GLUTs) which belong to family of over fourteen membrane proteins collectively known as the glucose GLUT/SLC2A family of glucose/ polyol transporters (Joost and Thorens, 2001; Joost et al., 2002; Wu and Freeze, 2002). Recent reports suggest that chondrocytes express multiple isoforms of the GLUT/SLC2A family, including the GLUT1 and GLUT3 proteins (Ohara et al., 2001; Shikhman et al., 2001a; Mobasheri et al., 2002a). Anabolic endocrine factors such as insulin-like growth factor I (IGF-I) stimulate glucose uptake in human chondrocytes (Richardson et al., 2003). Studies from our group (Richardson et al., 2003) and one other laboratory (Shikhman et al., 2001a) suggest that net glucose transport in human chondrocytes is also stimulated by pro-inflammatory cytokines such as interleukin 1 beta (IL-1b), interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-a). There is also evidence to suggest that the GLUT1 protein is a stress inducible glucose transporter in human chondrocytes and is significantly upregulated in cells under the influence of proinflammatory cytokines (Shikhman et al., 2001a). Therefore, enhanced glucose transport and glycolysis by articular chondrocytes may be important pre-requisites for cartilage degradation in degenerative diseases of articulating joints. However, it is not yet known if the individual GLUT isoforms expressed in equine articular chondrocytes respond to growth factors and cytokines in a similar way to human chondrocytes. It is also not known if the stimulated glucose uptake in response to these physiologically relevant mediators is the result of de novo synthesis and upregulation of glucose transporters. The primary objective of this study was to test a novel hypothesis that may implicate chondrocyte glucose transporters in osteoarticular disorders thus establishing a link between chondrocyte bioenergetics, nutrient status and joint disease. The hypothesis tested was that glucose transporters in equine chondrocytes are differentially regulated by growth factors and pro-inflammatory cytokines whose pathophysiological and bioenergetic effects may exacerbate the phenotypic and molecular alterations observed in degenerative joint disease. Accordingly, the aim of the investigation was to determine the effects of IL-1b, TNF-a, IGF-I and transforming growth factor beta (TGF-b) on (1) the net glucose uptake capacity of equine articular chondrocytes in monolayer culture and (2) the expression of the GLUT1 and GLUT3 glucose transporter protein isoforms in equine articular chondrocytes in alginate culture.
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2. Materials and methods 2.1. Chemicals All chemicals used in this study were molecular biology grade and purchased from Sigma/Aldrich. 2-Deoxy3 D -[2, 6- H] glucose was from Amersham Biosciences, protein assay kits were obtained from Bio-Rad and antibodies were purchased from Chemicon International. 2.2. Cartilage source Normal equine articular cartilage was obtained from the stifle joints of three horses (age range: 5–21 years) euthanased for clinical reasons at the Philip Leverhulme Large Animal Hospital, University of Liverpool. The study was conducted with ethical approval in strict accordance with local guidelines (none of these three horses were euthanased for the purpose of this study). 2.3. Chondrocyte isolation and culture Equine cartilage shavings were rinsed with phosphate-buffered saline (PBS), cut into small slices and incubated overnight with type I collagenase (EC 3.4.24.3 from Clostridium histolyticum) (approximately 100 collagen digestion units mL1 ) in Dulbecco’s modified Eagles medium (DMEM) supplemented with 1000 mg L1 glucose, 10% fetal calf serum and 1% antibiotic/ antimycotic solution. The filtered cell suspension was washed three times in fresh DMEM and the cells counted on a haemocytometer; cell viability was determined by trypan blue dye exclusion and was usually 95% or higher. The cells (2 106 cells/mL) were cultured in alginate beads as previously described (Mobasheri et al., 2002a; Richardson et al., 2003). Two different culture protocols were used in this study for the following reason: 2-deoxy-D -[2,6-3 H] glucose uptake experiments can only be performed with monolayer cultured chondrocytes as the final washes in non-radioactive medium cannot be performed rapidly with alginate beads. Cells used for uptake studies were not passaged more than three times to prevent chondrocyte dedifferentiation and phenotypic instability. Chondrocytes encapsulated and maintained in alginate culture were used for Western blot analyses as this method allows investigators to maintain equal cell numbers thus ensuring equal protein loading on SDS– PAGE. 2.4. Deoxy-D -[2,6-3 H] glucose uptake Net glucose transport was determined by measuring the uptake of non-metabolizable 2-deoxy-D -[2,6-3 H] glucose into equine chondrocytes in 24-well plates in the presence and absence of the glucose transport inhibitor
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cytochalasin B (1 lM). Most of the uptake experiments described were performed with chondrocytes stimulated for 24 h with TNF-a (100 ng mL1 ), IL-1b (100 ng mL1 ), IGF-I (20 ng mL1 ), TGF-b (20 ng mL1 ) and insulin (12.5 lg mL1 ). Selected experiments were carried to study the short-term effect of insulin on 2deoxyglucose uptake by pre-incubating chondrocytes for 15 min with insulin (12.5 lg mL1 ). The insulin used in this study was derived from the bovine species. All the cytokines and growth factors used in this study were human recombinants. Prior to the commencement of the assay, the chondrocyte cultures were rinsed three times with PBS and the uptake of the radiolabelled 2-deoxyglucose (1 lCi mL1 culture medium) was assayed for 35 min at 20 °C in modified DMEM lacking glucose, pyruvate and serum. The 24-well plates were washed three times with ice-cold PBS and the chondrocytes lysed with a cell lysis solution consisting of 0.5% sodium dodecyl sulphate and 0.5% Triton X-100 in PBS. Aliquots (450 lL) of the cell lysis solution were mixed with 3.55 mL of NACS104 aqueous scintillation cocktail (Amersham Biosciences) and counted in a scintillation counter. The remaining 50 lL aliquots of the cell lysis solution were used to determine total cell protein content using a Bio-Rad detergent compatible (DC) protein assay kit and 2-deoxyglucose uptake was normalized to total cell protein. All uptake experiments were carried out in triplicate and repeated under identical conditions at least three times and the data are presented as percentage change in total 2-deoxy3 D -[2,6- H] glucose uptake. 2.5. SDS–PAGE and western blot analysis Briefly, chondrocytes from alginate cultures were rinsed in PBS and proteins were extracted with lysis buffer (50 mM Tris/HCl, pH 7.2, 150 mM NaCl, l% (v/v) Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 0.01% (v/v) aprotinin, 4 lg/mL pepstatin A, 10 lg/mL leupeptin, 1 mM PMSF) on ice for 30 min. After adjustment of total protein concentration using a Bio-Rad DC protein assay kit, samples were separated by SDS–PAGE on 12% minigels under reducing conditions. Separated proteins were transferred onto nitrocellulose membranes (Sigma) and incubated in blocking buffer (5% (w/v) skimmed milk powder in PBS/0.1% Tween 20) for 1 h at room temperature (RT). Membranes were incubated with polyclonal primary antibodies to GLUT1 and GLUT3 (Chemicon International) diluted 1 in 1000 in blocking buffer for 1 h at RT, washed three times with blocking buffer, and incubated with secondary goat antirabbit antibody conjugated with alkaline phosphatase for 30 min at RT. Membranes were rinsed in blocking buffer and washed three times in 0.1 M Tris pH 9.5, containing 0.05 M MgCl2 and 0.1 M NaCl. Specific an-
tigen-antibody complexes were visualised by incubating with premixed nitro blue tetrazolium (NBT) in conjunction with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as precipitating agent for alkaline phosphatase. 2.6. Statistical analysis The results are expressed as the means SE of a representative experiment performed in triplicate. The means were compared using Student’s t test assuming equal variances. P < 0:05 was considered statistically significant. 2.7. Image analysis Quantification of Western blots was carried out using Scion Image for Windows (version 4.0.2 http://www. scioncorp.com/) based on NIH Image for Macintosh. The GLUT1 and GLUT3 immunoblots were scanned in 128 shades of grey. Thresholding was used to segment scanned blots into objects of interest displayed in black (i.e. GLUT bands) and background displayed in white based on the grey levels in the original blot.
3. Results We found that phenotypically stable equine articular chondrocytes were capable of transporting 2-deoxy-D [2,6-3 H] glucose, which is consistent with our previous observations in human chondrocytes and provides, for the first time evidence for the presence of functional glucose transporters in equine articular chondrocytes. 3.1. 2-Deoxy-D -[2,6-3 H] glucose uptake is not significantly influenced by insulin Equine chondrocytes in 24-well plates were stimulated with bovine insulin for up to 15 min (short term) at room temperature or at least 24 h (long term) at 37 °C before measuring net 2-deoxy-D -[2,6-3 H] glucose uptake. Uptake into untreated (control) chondrocytes was considered to be 100%. The specificity of uptake was determined by addition of cytochalasin B at the point of assay initiation. We found that short-term or long-term stimulation with bovine insulin did not affect 2-deoxyglucose uptake in a statistically significant manner (Fig. 1). Cytochalasin B inhibited 2-deoxyglucose transport in control and insulin stimulated cultures (P < 0:025). Short-term stimulation with insulin (15 min) resulted in a small but statistically insignificant (P ¼ 0:22) increase in net 2deoxy-D -[2,6-3 H] glucose uptake. Long-term incubation with insulin (24 h) also had no stimulatory effect on glucose uptake (P ¼ 0:15). These observations suggest that the insulin-responsive GLUT4 isoform is either not expressed in equine articular chondrocytes or that its
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P = 0.22
150
P = 0.15
125
100
3
% of 2-Deoxy-D-[2,6- H] Glucose Uptake
T. Phillips et al. / The Veterinary Journal 169 (2005) 216–222
75
*
50
*
*
25
0 Control
Control + Cyt B
Insulin (Short Term)
Insulin (Short Term) + Cyt B
Insulin (Long Term)
Insulin (Long Term) + Cyt B
Fig. 1. 2-Deoxy-D -[2,6-3 H] glucose uptake is not significantly influenced by insulin stimulation of equine chondrocytes. Cells were stimulated with insulin for 15 min (short term) at room temperature or 24 h (long term) at 37 °C. 2-deoxyglucose uptake into untreated (control) cells was considered to be 100%. Specificity of uptake was determined cytochalasin B treatment during the assay period. Neither short-term nor long-term stimulation with insulin resulted in a significant change in 2-deoxyglucose uptake. There was no significant increase in 2-deoxyglucose uptake following short term insulin stimulation (P ¼ 0:22). Long-term stimulation with insulin resulted in a decrease in 2-deoxyglucose uptake (P ¼ 0:15). * Denotes a significant difference (P < 0:025) between control and experimental groups.
plasma membrane abundance is too low to affect net glucose uptake in response to insulin treatment in equine articular chondrocytes. 3.2. Cytokine and endocrine stimulation of 2-deoxy-[2,63 H] glucose uptake Uptake of 2-deoxy-[2,6-3 H] glucose was significantly increased in equine articular chondrocytes pre-stimulated with IGF-I, TGF-b, TNF-a and IL-1b (Fig. 2). In these experiments baseline 2-deoxyglucose uptake was measured in un-stimulated (control) chondrocytes and
the value represented as 100%. Treatment with IGF-I, TGF-b, IL-1b and TNF-a resulted in increased 2-deoxyglucose uptake by over 65% compared to control values (P < 0:025). 3.3. GLUT1 expression is upregulated by pro-inflammatory cytokines and growth factors but only IGF-I upregulates GLUT3 We observed that the effects of growth factors and pro-inflammatory cytokines on the individual GLUT protein isoforms (i.e. GLUT1 and GLUT3) was not the
Fig. 2. Uptake of 2-deoxy-[2,6-3 H] glucose by equine articular chondrocytes stimulated with IGF-I, TGF-b, TNF-a and IL-1b. Baseline 2-deoxyglucose uptake in un-stimulated chondrocytes (control cells) was considered as 100%. Error bars indicate standard errors of the means (n ¼ 3). * Denotes a significant difference between control and experimental groups (P < 0:025).
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same despite the fact that the uptake of 2-deoxy-[2,6-3 H] glucose was significantly increased in equine articular chondrocytes pre-stimulated with IGF-I, TGF-b, TNFa and IL-1b. Western blot analysis of equine chondrocyte cell lysates demonstrated that equine chondrocytes expressed the GLUT1 and GLUT3 proteins with apparent molecular weights of between 55 and 60 kDa. Previous studies have revealed similar molecular weights for GLUT1 and GLUT3 in human chondrocytes. Stimulation of alginate cultures of equine chondrocytes with IGF-I, TGF-b, TNF-a and IL-1b all resulted in upregulation of GLUT1 (P < 0:05). In the case of GLUT3, a similar upregulation was measured for cultures exposed to IGF-I (P < 0:05). However TGF-b appeared to reduce the levels of GLUT3 protein and TNF-a and IL-1b had no significant effect on the abundance of GLUT3. Note that the same cells exhibited a large increase in 2-deoxyglucose uptake in response to TGF-b treatment which suggests that while GLUT3 was down-regulated in response to this growth factor, GLUT1 (and possibly other GLUT isoforms) may have been activated or significantly up-regulated, resulting in an increase in net 2-deoxyglucose transport.
4. Discussion The major findings of this study are summarized as follows: (1) equine chondrocytes express functional glucose transporters; (2) the uptake of 2-deoxy-D -[2,6-3 H] glucose into equine chondrocytes is consistent with a facilitated, GLUT-mediated process for glucose transport; (3) short-term insulin treatment resulted in a statistically insignificant increase in the uptake of 2-deoxyglucose but long-term insulin treatment had no effect on 2-deoxyglucose uptake; (4) IGF-I, IL-1b, TGF-b and TNF-a all resulted in a significant (over 65%) increase in glucose uptake compared to basal levels; (5) IGF-I stimulation of equine chondrocytes over a period of 24 h resulted in upregulation of GLUT1 and GLUT3 proteins; (6) TGF-b apparently reduced the levels of GLUT3; (7) TNF-a and IL-1b stimulation upregulated GLUT1 expression but down-regulated GLUT3 supporting the hypothesis that growth factors and pro-inflammatory cytokines differentially regulate the GLUT1 and GLUT3 glucose transporters. Articular cartilage is an avascular tissue in which oxygen tensions are generally very low (Otte, 1991; Mobasheri et al., 2002b). In the absence of oxidative phosphorylation, chondrocytes endure hypoxic conditions by generating ATP by glycolytic breakdown of glucose (Mobasheri et al., 2002a). Hypoxia generally induces expression of several important genes including vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT1; SLC2A1) and glucose transporter-3 (GLUT3; SLC2A3) (Semenza, 1999). Evidence
from other cell types in the literature suggests that elevated glucose uptake stimulated by pro-inflammatory cytokines is accompanied by upregulation of GLUT transporter proteins. In response to pro-inflammatory cytokines a number of genes are activated including VEGF and GLUT1 (Pufe et al., 2001; Shikhman et al., 2001a; Richardson et al., 2003) resulting in the upregulation of the proteins encoded by these genes in chondrocytes. These proteins are key mediators of nutrient and energy provision in degenerative conditions such as osteoarthritis, which is particularly relevant to the equine species; the glucose transporters provide the means for accelerated glucose transport and enhanced substrate utilization in response to stimulation by pro-inflammatory, catabolic cytokines, including TNF-a, IL-6 and IL1b (Shikhman et al., 2001a; Shikhman et al., 2001b; Richardson et al., 2003) and to chemokines such as connective tissue activating peptide III (Ku Tai et al., 1992). Studies in chondrocytes from the growth plates of rodents (Wang et al., 1999; Ohara et al., 2001) and in human Meckel’s cartilage (Ishizeki et al., 2002) have shown that the insulin-responsive GLUT4 isoform is expressed and functional in these cells. However, we and others have previously reported on the absence of insulin-responsive glucose transporters in mature human articular chondrocytes (Shikhman et al., 2001a; Mobasheri et al., 2002a). The results presented in this study (Fig. 1) support our earlier observations in human chondrocytes (Richardson et al., 2003) and confirm that insulin-responsive glucose transporters are not expressed in fully developed equine articular chondrocytes, and that insulin does not acutely increase glucose transport in these cells. In the presence of high levels of serum, chondrocytes from Meckel’s cartilage have been shown to change phenotype and differentiate into adipocytes (Ishizeki et al., 2002). There are no published reports that suggest articular chondrocytes are capable of undergoing a similar transdifferentiation. The cytokines and growth factors used in this study were human recombinants. This was due to the absence of suitable equine counterparts. The observed differences in the magnitude of cytokine and growth factor stimulated 2-deoxyglucose transport (Fig. 2) is likely to be due to the lower biological activity of human recombinant cytokines and growth factors on equine chondrocytes. The magnitude of the changes we observed in glucose uptake in response to IGF-I, IL-1b and TNF-a was similar to the values previously reported by our group (Richardson et al., 2003). However, the augmented 2-deoxyglucose uptake was lower than values reported by another group for human chondrocytes (Shikhman et al., 2001a). In response to long-term TGF-b stimulation we observed increased 2-deoxyglucose transport and upregulation of GLUT1 but GLUT3 appeared to be down-regulated (Fig. 3). Studies in the kidney have
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Fig. 3. Evidence for growth factor and cytokine regulation of GLUT1 and GLUT3 expression in equine articular chondrocytes. Western blot analysis demonstrated that equine chondrocytes expressed GLUT1 and GLUT3. IGF-I stimulation resulted in upregulation of GLUT1 and GLUT3. TGF-b stimulation upregulated GLUT1 but down-regulated GLUT3. TNF-a and IL-1b stimulation resulted in upregulation of GLUT1 but did not affect GLUT3. TGF-b also reduced the levels of the GLUT3 protein. * Denotes a significant difference (P < 0:05); ns denotes a statistically insignificant difference.
shown that TGF-b stimulates glucose transport by enhancing GLUT1 expression in mesangial cells (Inoki et al., 1999), which confirms our observations. TGF-b also stimulates glucose transport by increasing GLUT1 expression in fibroblasts (Kitagawa et al., 1991). The expression of GLUT3 has not been studied in response to TGF-b stimulation (no published reports in PubMed) making it difficult to compare our results with published information in the literature. Elevated glucose transport and upregulation of GLUT proteins may be an early and sustained event in inflammatory processes in equine articular cartilage. The increased glucose uptake observed in equine chondrocytes stimulated with pro-inflammatory cytokines and growth factors appears to be an important component of the chondrocyte response to both anabolic and catabolic mediators and is likely to be due to the mutual dependency of catabolic and anabolic pathways on regulated glucose transport (Mobasheri et al., 2002b). However, the GLUT isoforms appear to respond differently to different stimuli. GLUT2 and GLUT4 proteins have not been detected by immunohistochemical methods in equine articular cartilage (A. Mobasheri, unpublished observations) supporting previous findings in human chondrocytes (Shikhman et al., 2001a; Mobasheri et al., 2002a). Further studies are needed to determine if other GLUT isoforms identified in chondrocytes (i.e. GLUT5, GLUT9 and GLUT12) (Ohara et al., 2001; Shikhman et al., 2001a; Macheda et al., 2002; Richardson et al., 2003) are involved in the chondrocyte responses to growth factors and cytokines.
Arthropathies are a major cause of poor welfare in the horse and the physiology and nutrition of articular cartilage are key issues in this process. Cartilage degradation in equine osteoarthritis has been shown to be stimulated by pro-inflammatory cytokines and accompanied by apoptosis (Clegg and Mobasheri, 2003; Kim et al., 2003) and elevated matrix metalloprotease secretion (particularly MMP-2 and MMP-9; Clegg and Carter, 1999). The data presented here suggests that growth factors can stimulate net glucose uptake by chondrocytes. Furthermore, pro-inflammatory cytokines also stimulate increased uptake of glucose in order to provide the metabolic energy required for extracellular matrix degradation. However, growth factors and pro-inflammatory cytokines have differential effects on the individual GLUT isoforms; most up-regulate GLUT1 expression but GLUT3 is only up-regulated by IGF-I. A clearer understanding of cytokine and endocrine regulated transport systems in chondrocyte physiology may lead to identification of key molecules or biochemical pathways involved in the nutritional regulation of cartilage matrix integrity.
Acknowledgements We would like to thank the University of Liverpool Research Development Fund for funding this work. T. Phillips would like to acknowledge the support of Professor David J. Back (Department of Pharmacology and Therapeutics, University of Liverpool). This study was
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also funded by a short-term fellowship granted to I. Ferraz from the Servicio de Reumatologia, Hospital Universitario de Canarias, in Santa Cruz de Tenerife. References Clegg, P.D., Carter, S.D., 1999. Matrix metalloproteinase-2 and -9 are activated in joint diseases. Equine Veterinary Journal 31, 324–330. Clegg, P.D., Mobasheri, A., 2003. Chondrocyte apoptosis, inflammatory mediators and equine osteoarthritis. The Veterinary Journal 166, 3–4. Inoki, K., Haneda, M., Maeda, S., Koya, D., Kikkawa, R., 1999. TGF-beta 1 stimulates glucose uptake by enhancing GLUT1 expression in mesangial cells. Kidney International 55, 1704–1712. Ishizeki, K., Takahashi, N., Nawa, T., 2002. Phenotypic characteristics of adipocytes generated from Meckel’s chondrocytes in response to chick serum in vitro. Cell and Tissue Research 309, 251–260. Joost, H.G., Thorens, B., 2001. The extended GLUT-family of sugar/ polyol transport facilitators: nomenclature, sequence characteristics, and potential function of its novel members (review). Molecular Membrane Biology 18, 247–256. Joost, H.G., Bell, G.I., Best, J.D., Birnbaum, M.J., Charron, M.J., Chen, Y.T., Doege, H., James, D.E., Lodish, H.F., Moley, K.H., Moley, J.F., Mueckler, M., Rogers, S., Schurmann, A., Seino, S., Thorens, B., 2002. Nomenclature of the GLUT/SLC2A family of sugar/polyol transport facilitators. American Journal of Physiology Endocrinology and Metabolism 282, E974–E976. Kim, D.Y., Taylor, H.W., Moore, R.M., Paulsen, D.B., Cho, D.Y., 2003. Articular chondrocyte apoptosis in equine osteoarthritis. The Veterinary Journal 166, 52–57. Kitagawa, T., Masumi, A., Akamatsu, Y., 1991. Transforming growth factor-beta 1 stimulates glucose uptake and the expression of glucose transporter mRNA in quiescent Swiss mouse 3T3 cells. Journal of Biological Chemistry 266, 18066–18071. Ku Tai, P.-K., Liao, J.-F., Hossler, P.A., Castor, C.W., Carter-Su, C., 1992. Regulation of glucose transporters by connective tissue activating peptide-III isoforms. Journal of Biological Chemistry 267, 19579–19586. Macheda, M.L., Kelly, D.J., Best, J.D., Rogers, S., 2002. Expression during rat fetal development of GLUT12-a member of the class III hexose transporter family. Anatomy and Embryology (Berlin) 205, 441–452.
Mobasheri, A., Neama, G., Bell, S., Richardson, S., Carter, S.D., 2002a. Human articular chondrocytes express three facilitative glucose transporter isoforms: GLUT1, GLUT3 and GLUT9. Cell Biology International 26, 297–300. Mobasheri, A., Vannucci, S.J., Bondy, C.A., Carter, S.D., Innes, J.F., Arteaga, M.F., Trujillo, E., Ferraz, I., Shakibaei, M., MartinVasallo, P., 2002b. Glucose transport and metabolism in chondrocytes: a key to understanding chondrogenesis, skeletal development and cartilage degradation in osteoarthritis. Histology and Histopathology 17, 1239–1267. Ohara, H., Tamayama, T., Maemura, K., Kanbara, K., Hayasaki, H., Abe, M., Watanabe, M., 2001. Immunocytochemical demonstration of glucose transporters in epiphyseal growth plate chondrocytes of young rats in correlation with autoradiographic distribution of 2-deoxyglucose in chondrocytes of mice. Acta Histochemica 103, 365–378. Otte, P., 1991. Basic cell metabolism of articular cartilage. Manometric studies. Zeitschrift f€ ur Rheumatologie 50, 304–312. Pufe, T., Petersen, W., Tillmann, B., Mentlein, R., 2001. The splice variants VEGF121 and VEGF189 of the angiogenic peptide vascular endothelial growth factor are expressed in osteoarthritic cartilage. Arthritis and Rheumatism 44, 1082–1088. Richardson, S., Neama, G., Phillips, T., Bell, S., Carter, S.D., Moley, K.H., Moley, J.F., Vannucci, S.J., Mobasheri, A., 2003. Molecular characterization and partial cDNA cloning of facilitative glucose transporters expressed in human articular chondrocytes; stimulation of 2-deoxyglucose uptake by IGF-I and elevated MMP-2 secretion by glucose deprivation. Osteoarthritis and Cartilage 11, 92–101. Semenza, G.L., 1999. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annual Review of Cell and Developmental Biology 15, 551–578. Shikhman, A.R., Brinson, D.C., Valbracht, J., Lotz, M.K., 2001a. Cytokine regulation of facilitated glucose transport in human articular chondrocytes. Journal of Immunology 167, 7001–7008. Shikhman, A.R., Kuhn, K., Alaaeddine, N., Lotz, M., 2001b. Nacetylglucosamine prevents IL-1 beta-mediated activation of human chondrocytes. Journal of Immunology 166, 5155–5160. Wang, J., Zhou, J., Bondy, C.A., 1999. Igf1 promotes longitudinal bone growth by insulin-like actions augmenting chondrocyte hypertrophy. FASEB Journal 13, 1985–1990. Wu, X., Freeze, H.H., 2002. GLUT14, a duplicon of GLUT3, is specifically expressed in testis as alternative splice forms. Genomics 80, 553–557.
Differential regulation of the GLUT1 and GLUT3 glucose transporters by growth factors and pro-inflammatory cytokines in equine articular chondrocytes Toby Phillips, Ivan Ferraz 1, Susan Bell, Peter D. Clegg, Stuart D. Carter, Ali Mobasheri * Connective Tissue and Molecular Pathogenesis Research Groups, Faculty of Veterinary Science, University of Liverpool, Liverpool L69 7ZJ, UK Accepted 16 January 2004
Abstract Glucose serves as the major energy substrate for articular chondrocytes and as the main precursor for the synthesis of extracellular matrix glycosaminoglycans in cartilage. Chondrocytes have been shown to express several glucose transporter (GLUT) isoforms including GLUT1 and GLUT3. The aim of this investigation was to determine the effects of endocrine and cytokine factors on the capacity of equine articular chondrocytes for transporting 2-deoxy-D -[2, 6-3 H] glucose and on the expression levels of GLUT1 and GLUT3. Chondrocytes maintained in monolayer culture were stimulated for 24 h with TNF-a (100 ng mL1 ), IL-1b (100 ng mL1 ), IGF-I (20 ng mL1 ), TGF-b (20 ng mL1 ) and insulin (12.5 lg mL1 ) before measuring uptake of non-metabolizable 2-deoxyglucose in the presence and absence of the glucose transport inhibitor cytochalasin B. Polyclonal antibodies to GLUT1 and GLUT were used to compare GLUT1 and GLUT3 expression in stimulated and un-stimulated alginate encapsulated chondrocytes by Western blotting. Results indicated that 2-deoxyglucose uptake was inhibited by up to 95% in the presence of cytochalasin B suggesting that glucose uptake into equine chondrocytes is GLUT-mediated. Insulin had no effect on glucose uptake, but treatment with IGF-I, TGF-b, IL-1b and TNF-a resulted in a significant increase (>65%) in 2-deoxyglucose uptake compared to control values. GLUT1 was found to be increased in chondrocytes stimulated with all the growth factors and cytokines but GLUT 3 was only upregulated by IGF-I. The data presented support a critical role for glucose in the responses of equine articular chondrocytes to pro-inflammatory cytokines and anabolic endocrine factors. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Cartilage; Chondrocyte; Glucose transport; GLUT; Cytokine; Growth factor
1. Introduction Recent research suggests that osteoarticular disorders in humans and veterinary species may be directly linked to obesity and may therefore have nutritional and en-
*
Corresponding author. Tel.: +44-151-794-4284; fax: +44-151-7944243. E-mail address: [email protected] (A. Mobasheri). 1 Present Address: Servicio de Reumatologia, Hospital Universitario de Canarias, Tenerife, La Cuesta, Santa Cruz de Tenerife and Departamento de Bioquımica y Biologıa Molecular, Universidad de La Laguna, Tenerife, Spain. 1090-0233/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2004.01.026
docrine abnormalities at the root of their pathogenesis. Glucose is an essential energy source for mammalian cells and in articular cartilage glucose plays a pivotal role in the physiology of the chondrocytes by driving the extracellular matrix biosynthetic machinery of this unique cell type (Wang et al., 1999; Mobasheri et al., 2002b). Glucose is also a major energy source and a precursor for the synthesis of glycosaminoglycans (Mobasheri et al., 2002a). Despite this realisation, there is limited published information about the molecular mechanisms responsible for nutrient transport across the chondrocyte membrane and their regulation by growth factors and pro-inflammatory cytokines.
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The facilitated transport of glucose and glucosederived compounds across the chondrocyte membrane represents the rate-limiting step in glucose metabolism (Shikhman et al., 2001a) and is therefore essential for chondrogenesis and the functional integrity of articulating joints. Mammalian cells transport glucose across the plasma membrane by facilitated diffusion through the glucose transporters (or GLUTs) which belong to family of over fourteen membrane proteins collectively known as the glucose GLUT/SLC2A family of glucose/ polyol transporters (Joost and Thorens, 2001; Joost et al., 2002; Wu and Freeze, 2002). Recent reports suggest that chondrocytes express multiple isoforms of the GLUT/SLC2A family, including the GLUT1 and GLUT3 proteins (Ohara et al., 2001; Shikhman et al., 2001a; Mobasheri et al., 2002a). Anabolic endocrine factors such as insulin-like growth factor I (IGF-I) stimulate glucose uptake in human chondrocytes (Richardson et al., 2003). Studies from our group (Richardson et al., 2003) and one other laboratory (Shikhman et al., 2001a) suggest that net glucose transport in human chondrocytes is also stimulated by pro-inflammatory cytokines such as interleukin 1 beta (IL-1b), interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-a). There is also evidence to suggest that the GLUT1 protein is a stress inducible glucose transporter in human chondrocytes and is significantly upregulated in cells under the influence of proinflammatory cytokines (Shikhman et al., 2001a). Therefore, enhanced glucose transport and glycolysis by articular chondrocytes may be important pre-requisites for cartilage degradation in degenerative diseases of articulating joints. However, it is not yet known if the individual GLUT isoforms expressed in equine articular chondrocytes respond to growth factors and cytokines in a similar way to human chondrocytes. It is also not known if the stimulated glucose uptake in response to these physiologically relevant mediators is the result of de novo synthesis and upregulation of glucose transporters. The primary objective of this study was to test a novel hypothesis that may implicate chondrocyte glucose transporters in osteoarticular disorders thus establishing a link between chondrocyte bioenergetics, nutrient status and joint disease. The hypothesis tested was that glucose transporters in equine chondrocytes are differentially regulated by growth factors and pro-inflammatory cytokines whose pathophysiological and bioenergetic effects may exacerbate the phenotypic and molecular alterations observed in degenerative joint disease. Accordingly, the aim of the investigation was to determine the effects of IL-1b, TNF-a, IGF-I and transforming growth factor beta (TGF-b) on (1) the net glucose uptake capacity of equine articular chondrocytes in monolayer culture and (2) the expression of the GLUT1 and GLUT3 glucose transporter protein isoforms in equine articular chondrocytes in alginate culture.
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2. Materials and methods 2.1. Chemicals All chemicals used in this study were molecular biology grade and purchased from Sigma/Aldrich. 2-Deoxy3 D -[2, 6- H] glucose was from Amersham Biosciences, protein assay kits were obtained from Bio-Rad and antibodies were purchased from Chemicon International. 2.2. Cartilage source Normal equine articular cartilage was obtained from the stifle joints of three horses (age range: 5–21 years) euthanased for clinical reasons at the Philip Leverhulme Large Animal Hospital, University of Liverpool. The study was conducted with ethical approval in strict accordance with local guidelines (none of these three horses were euthanased for the purpose of this study). 2.3. Chondrocyte isolation and culture Equine cartilage shavings were rinsed with phosphate-buffered saline (PBS), cut into small slices and incubated overnight with type I collagenase (EC 3.4.24.3 from Clostridium histolyticum) (approximately 100 collagen digestion units mL1 ) in Dulbecco’s modified Eagles medium (DMEM) supplemented with 1000 mg L1 glucose, 10% fetal calf serum and 1% antibiotic/ antimycotic solution. The filtered cell suspension was washed three times in fresh DMEM and the cells counted on a haemocytometer; cell viability was determined by trypan blue dye exclusion and was usually 95% or higher. The cells (2 106 cells/mL) were cultured in alginate beads as previously described (Mobasheri et al., 2002a; Richardson et al., 2003). Two different culture protocols were used in this study for the following reason: 2-deoxy-D -[2,6-3 H] glucose uptake experiments can only be performed with monolayer cultured chondrocytes as the final washes in non-radioactive medium cannot be performed rapidly with alginate beads. Cells used for uptake studies were not passaged more than three times to prevent chondrocyte dedifferentiation and phenotypic instability. Chondrocytes encapsulated and maintained in alginate culture were used for Western blot analyses as this method allows investigators to maintain equal cell numbers thus ensuring equal protein loading on SDS– PAGE. 2.4. Deoxy-D -[2,6-3 H] glucose uptake Net glucose transport was determined by measuring the uptake of non-metabolizable 2-deoxy-D -[2,6-3 H] glucose into equine chondrocytes in 24-well plates in the presence and absence of the glucose transport inhibitor
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cytochalasin B (1 lM). Most of the uptake experiments described were performed with chondrocytes stimulated for 24 h with TNF-a (100 ng mL1 ), IL-1b (100 ng mL1 ), IGF-I (20 ng mL1 ), TGF-b (20 ng mL1 ) and insulin (12.5 lg mL1 ). Selected experiments were carried to study the short-term effect of insulin on 2deoxyglucose uptake by pre-incubating chondrocytes for 15 min with insulin (12.5 lg mL1 ). The insulin used in this study was derived from the bovine species. All the cytokines and growth factors used in this study were human recombinants. Prior to the commencement of the assay, the chondrocyte cultures were rinsed three times with PBS and the uptake of the radiolabelled 2-deoxyglucose (1 lCi mL1 culture medium) was assayed for 35 min at 20 °C in modified DMEM lacking glucose, pyruvate and serum. The 24-well plates were washed three times with ice-cold PBS and the chondrocytes lysed with a cell lysis solution consisting of 0.5% sodium dodecyl sulphate and 0.5% Triton X-100 in PBS. Aliquots (450 lL) of the cell lysis solution were mixed with 3.55 mL of NACS104 aqueous scintillation cocktail (Amersham Biosciences) and counted in a scintillation counter. The remaining 50 lL aliquots of the cell lysis solution were used to determine total cell protein content using a Bio-Rad detergent compatible (DC) protein assay kit and 2-deoxyglucose uptake was normalized to total cell protein. All uptake experiments were carried out in triplicate and repeated under identical conditions at least three times and the data are presented as percentage change in total 2-deoxy3 D -[2,6- H] glucose uptake. 2.5. SDS–PAGE and western blot analysis Briefly, chondrocytes from alginate cultures were rinsed in PBS and proteins were extracted with lysis buffer (50 mM Tris/HCl, pH 7.2, 150 mM NaCl, l% (v/v) Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 0.01% (v/v) aprotinin, 4 lg/mL pepstatin A, 10 lg/mL leupeptin, 1 mM PMSF) on ice for 30 min. After adjustment of total protein concentration using a Bio-Rad DC protein assay kit, samples were separated by SDS–PAGE on 12% minigels under reducing conditions. Separated proteins were transferred onto nitrocellulose membranes (Sigma) and incubated in blocking buffer (5% (w/v) skimmed milk powder in PBS/0.1% Tween 20) for 1 h at room temperature (RT). Membranes were incubated with polyclonal primary antibodies to GLUT1 and GLUT3 (Chemicon International) diluted 1 in 1000 in blocking buffer for 1 h at RT, washed three times with blocking buffer, and incubated with secondary goat antirabbit antibody conjugated with alkaline phosphatase for 30 min at RT. Membranes were rinsed in blocking buffer and washed three times in 0.1 M Tris pH 9.5, containing 0.05 M MgCl2 and 0.1 M NaCl. Specific an-
tigen-antibody complexes were visualised by incubating with premixed nitro blue tetrazolium (NBT) in conjunction with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as precipitating agent for alkaline phosphatase. 2.6. Statistical analysis The results are expressed as the means SE of a representative experiment performed in triplicate. The means were compared using Student’s t test assuming equal variances. P < 0:05 was considered statistically significant. 2.7. Image analysis Quantification of Western blots was carried out using Scion Image for Windows (version 4.0.2 http://www. scioncorp.com/) based on NIH Image for Macintosh. The GLUT1 and GLUT3 immunoblots were scanned in 128 shades of grey. Thresholding was used to segment scanned blots into objects of interest displayed in black (i.e. GLUT bands) and background displayed in white based on the grey levels in the original blot.
3. Results We found that phenotypically stable equine articular chondrocytes were capable of transporting 2-deoxy-D [2,6-3 H] glucose, which is consistent with our previous observations in human chondrocytes and provides, for the first time evidence for the presence of functional glucose transporters in equine articular chondrocytes. 3.1. 2-Deoxy-D -[2,6-3 H] glucose uptake is not significantly influenced by insulin Equine chondrocytes in 24-well plates were stimulated with bovine insulin for up to 15 min (short term) at room temperature or at least 24 h (long term) at 37 °C before measuring net 2-deoxy-D -[2,6-3 H] glucose uptake. Uptake into untreated (control) chondrocytes was considered to be 100%. The specificity of uptake was determined by addition of cytochalasin B at the point of assay initiation. We found that short-term or long-term stimulation with bovine insulin did not affect 2-deoxyglucose uptake in a statistically significant manner (Fig. 1). Cytochalasin B inhibited 2-deoxyglucose transport in control and insulin stimulated cultures (P < 0:025). Short-term stimulation with insulin (15 min) resulted in a small but statistically insignificant (P ¼ 0:22) increase in net 2deoxy-D -[2,6-3 H] glucose uptake. Long-term incubation with insulin (24 h) also had no stimulatory effect on glucose uptake (P ¼ 0:15). These observations suggest that the insulin-responsive GLUT4 isoform is either not expressed in equine articular chondrocytes or that its
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P = 0.22
150
P = 0.15
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3
% of 2-Deoxy-D-[2,6- H] Glucose Uptake
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75
*
50
*
*
25
0 Control
Control + Cyt B
Insulin (Short Term)
Insulin (Short Term) + Cyt B
Insulin (Long Term)
Insulin (Long Term) + Cyt B
Fig. 1. 2-Deoxy-D -[2,6-3 H] glucose uptake is not significantly influenced by insulin stimulation of equine chondrocytes. Cells were stimulated with insulin for 15 min (short term) at room temperature or 24 h (long term) at 37 °C. 2-deoxyglucose uptake into untreated (control) cells was considered to be 100%. Specificity of uptake was determined cytochalasin B treatment during the assay period. Neither short-term nor long-term stimulation with insulin resulted in a significant change in 2-deoxyglucose uptake. There was no significant increase in 2-deoxyglucose uptake following short term insulin stimulation (P ¼ 0:22). Long-term stimulation with insulin resulted in a decrease in 2-deoxyglucose uptake (P ¼ 0:15). * Denotes a significant difference (P < 0:025) between control and experimental groups.
plasma membrane abundance is too low to affect net glucose uptake in response to insulin treatment in equine articular chondrocytes. 3.2. Cytokine and endocrine stimulation of 2-deoxy-[2,63 H] glucose uptake Uptake of 2-deoxy-[2,6-3 H] glucose was significantly increased in equine articular chondrocytes pre-stimulated with IGF-I, TGF-b, TNF-a and IL-1b (Fig. 2). In these experiments baseline 2-deoxyglucose uptake was measured in un-stimulated (control) chondrocytes and
the value represented as 100%. Treatment with IGF-I, TGF-b, IL-1b and TNF-a resulted in increased 2-deoxyglucose uptake by over 65% compared to control values (P < 0:025). 3.3. GLUT1 expression is upregulated by pro-inflammatory cytokines and growth factors but only IGF-I upregulates GLUT3 We observed that the effects of growth factors and pro-inflammatory cytokines on the individual GLUT protein isoforms (i.e. GLUT1 and GLUT3) was not the
Fig. 2. Uptake of 2-deoxy-[2,6-3 H] glucose by equine articular chondrocytes stimulated with IGF-I, TGF-b, TNF-a and IL-1b. Baseline 2-deoxyglucose uptake in un-stimulated chondrocytes (control cells) was considered as 100%. Error bars indicate standard errors of the means (n ¼ 3). * Denotes a significant difference between control and experimental groups (P < 0:025).
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same despite the fact that the uptake of 2-deoxy-[2,6-3 H] glucose was significantly increased in equine articular chondrocytes pre-stimulated with IGF-I, TGF-b, TNFa and IL-1b. Western blot analysis of equine chondrocyte cell lysates demonstrated that equine chondrocytes expressed the GLUT1 and GLUT3 proteins with apparent molecular weights of between 55 and 60 kDa. Previous studies have revealed similar molecular weights for GLUT1 and GLUT3 in human chondrocytes. Stimulation of alginate cultures of equine chondrocytes with IGF-I, TGF-b, TNF-a and IL-1b all resulted in upregulation of GLUT1 (P < 0:05). In the case of GLUT3, a similar upregulation was measured for cultures exposed to IGF-I (P < 0:05). However TGF-b appeared to reduce the levels of GLUT3 protein and TNF-a and IL-1b had no significant effect on the abundance of GLUT3. Note that the same cells exhibited a large increase in 2-deoxyglucose uptake in response to TGF-b treatment which suggests that while GLUT3 was down-regulated in response to this growth factor, GLUT1 (and possibly other GLUT isoforms) may have been activated or significantly up-regulated, resulting in an increase in net 2-deoxyglucose transport.
4. Discussion The major findings of this study are summarized as follows: (1) equine chondrocytes express functional glucose transporters; (2) the uptake of 2-deoxy-D -[2,6-3 H] glucose into equine chondrocytes is consistent with a facilitated, GLUT-mediated process for glucose transport; (3) short-term insulin treatment resulted in a statistically insignificant increase in the uptake of 2-deoxyglucose but long-term insulin treatment had no effect on 2-deoxyglucose uptake; (4) IGF-I, IL-1b, TGF-b and TNF-a all resulted in a significant (over 65%) increase in glucose uptake compared to basal levels; (5) IGF-I stimulation of equine chondrocytes over a period of 24 h resulted in upregulation of GLUT1 and GLUT3 proteins; (6) TGF-b apparently reduced the levels of GLUT3; (7) TNF-a and IL-1b stimulation upregulated GLUT1 expression but down-regulated GLUT3 supporting the hypothesis that growth factors and pro-inflammatory cytokines differentially regulate the GLUT1 and GLUT3 glucose transporters. Articular cartilage is an avascular tissue in which oxygen tensions are generally very low (Otte, 1991; Mobasheri et al., 2002b). In the absence of oxidative phosphorylation, chondrocytes endure hypoxic conditions by generating ATP by glycolytic breakdown of glucose (Mobasheri et al., 2002a). Hypoxia generally induces expression of several important genes including vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT1; SLC2A1) and glucose transporter-3 (GLUT3; SLC2A3) (Semenza, 1999). Evidence
from other cell types in the literature suggests that elevated glucose uptake stimulated by pro-inflammatory cytokines is accompanied by upregulation of GLUT transporter proteins. In response to pro-inflammatory cytokines a number of genes are activated including VEGF and GLUT1 (Pufe et al., 2001; Shikhman et al., 2001a; Richardson et al., 2003) resulting in the upregulation of the proteins encoded by these genes in chondrocytes. These proteins are key mediators of nutrient and energy provision in degenerative conditions such as osteoarthritis, which is particularly relevant to the equine species; the glucose transporters provide the means for accelerated glucose transport and enhanced substrate utilization in response to stimulation by pro-inflammatory, catabolic cytokines, including TNF-a, IL-6 and IL1b (Shikhman et al., 2001a; Shikhman et al., 2001b; Richardson et al., 2003) and to chemokines such as connective tissue activating peptide III (Ku Tai et al., 1992). Studies in chondrocytes from the growth plates of rodents (Wang et al., 1999; Ohara et al., 2001) and in human Meckel’s cartilage (Ishizeki et al., 2002) have shown that the insulin-responsive GLUT4 isoform is expressed and functional in these cells. However, we and others have previously reported on the absence of insulin-responsive glucose transporters in mature human articular chondrocytes (Shikhman et al., 2001a; Mobasheri et al., 2002a). The results presented in this study (Fig. 1) support our earlier observations in human chondrocytes (Richardson et al., 2003) and confirm that insulin-responsive glucose transporters are not expressed in fully developed equine articular chondrocytes, and that insulin does not acutely increase glucose transport in these cells. In the presence of high levels of serum, chondrocytes from Meckel’s cartilage have been shown to change phenotype and differentiate into adipocytes (Ishizeki et al., 2002). There are no published reports that suggest articular chondrocytes are capable of undergoing a similar transdifferentiation. The cytokines and growth factors used in this study were human recombinants. This was due to the absence of suitable equine counterparts. The observed differences in the magnitude of cytokine and growth factor stimulated 2-deoxyglucose transport (Fig. 2) is likely to be due to the lower biological activity of human recombinant cytokines and growth factors on equine chondrocytes. The magnitude of the changes we observed in glucose uptake in response to IGF-I, IL-1b and TNF-a was similar to the values previously reported by our group (Richardson et al., 2003). However, the augmented 2-deoxyglucose uptake was lower than values reported by another group for human chondrocytes (Shikhman et al., 2001a). In response to long-term TGF-b stimulation we observed increased 2-deoxyglucose transport and upregulation of GLUT1 but GLUT3 appeared to be down-regulated (Fig. 3). Studies in the kidney have
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Fig. 3. Evidence for growth factor and cytokine regulation of GLUT1 and GLUT3 expression in equine articular chondrocytes. Western blot analysis demonstrated that equine chondrocytes expressed GLUT1 and GLUT3. IGF-I stimulation resulted in upregulation of GLUT1 and GLUT3. TGF-b stimulation upregulated GLUT1 but down-regulated GLUT3. TNF-a and IL-1b stimulation resulted in upregulation of GLUT1 but did not affect GLUT3. TGF-b also reduced the levels of the GLUT3 protein. * Denotes a significant difference (P < 0:05); ns denotes a statistically insignificant difference.
shown that TGF-b stimulates glucose transport by enhancing GLUT1 expression in mesangial cells (Inoki et al., 1999), which confirms our observations. TGF-b also stimulates glucose transport by increasing GLUT1 expression in fibroblasts (Kitagawa et al., 1991). The expression of GLUT3 has not been studied in response to TGF-b stimulation (no published reports in PubMed) making it difficult to compare our results with published information in the literature. Elevated glucose transport and upregulation of GLUT proteins may be an early and sustained event in inflammatory processes in equine articular cartilage. The increased glucose uptake observed in equine chondrocytes stimulated with pro-inflammatory cytokines and growth factors appears to be an important component of the chondrocyte response to both anabolic and catabolic mediators and is likely to be due to the mutual dependency of catabolic and anabolic pathways on regulated glucose transport (Mobasheri et al., 2002b). However, the GLUT isoforms appear to respond differently to different stimuli. GLUT2 and GLUT4 proteins have not been detected by immunohistochemical methods in equine articular cartilage (A. Mobasheri, unpublished observations) supporting previous findings in human chondrocytes (Shikhman et al., 2001a; Mobasheri et al., 2002a). Further studies are needed to determine if other GLUT isoforms identified in chondrocytes (i.e. GLUT5, GLUT9 and GLUT12) (Ohara et al., 2001; Shikhman et al., 2001a; Macheda et al., 2002; Richardson et al., 2003) are involved in the chondrocyte responses to growth factors and cytokines.
Arthropathies are a major cause of poor welfare in the horse and the physiology and nutrition of articular cartilage are key issues in this process. Cartilage degradation in equine osteoarthritis has been shown to be stimulated by pro-inflammatory cytokines and accompanied by apoptosis (Clegg and Mobasheri, 2003; Kim et al., 2003) and elevated matrix metalloprotease secretion (particularly MMP-2 and MMP-9; Clegg and Carter, 1999). The data presented here suggests that growth factors can stimulate net glucose uptake by chondrocytes. Furthermore, pro-inflammatory cytokines also stimulate increased uptake of glucose in order to provide the metabolic energy required for extracellular matrix degradation. However, growth factors and pro-inflammatory cytokines have differential effects on the individual GLUT isoforms; most up-regulate GLUT1 expression but GLUT3 is only up-regulated by IGF-I. A clearer understanding of cytokine and endocrine regulated transport systems in chondrocyte physiology may lead to identification of key molecules or biochemical pathways involved in the nutritional regulation of cartilage matrix integrity.
Acknowledgements We would like to thank the University of Liverpool Research Development Fund for funding this work. T. Phillips would like to acknowledge the support of Professor David J. Back (Department of Pharmacology and Therapeutics, University of Liverpool). This study was
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