The transport of nutrients through articular cartilage is critical for the viability and structural integrity of the articulating joint. Glucose is not only a structural precursor for the synthesis of extracellular matrix macromolecules, but also, it is an essential nutrient that must diffuse across the synovium and the extracellular matrix before reaching chondrocytes (Torzilli et al. 1997). Chondrocytes are highly glycolytic cells that exhibit negative Pasteur effects and require a regular supply of glucose for optimal ATP production and cell homeostasis. Thus far, the molecular identity of glucose transporters in articular chondrocytes has remained unexplored and the aim of this study was to identify glucose transporters present in normal human articular cartilage and human chondrocyte-like cells at the mRNA and protein levels.

Immortalized human chondrocytes (C20-A4) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, 280 mosmol l^-1) containing 10 % FCS, 1 % antibiotic/ antimycotic solution as monolayer and alginate cultures maintained at 37°C, 5 % CO₂ (Guo et al. 1989). Studies on human knee and hip cartilage derived from patients undergoing joint replacement surgery were carried out with ethical permission and informed consent. We used human specific oligonucleotide primers designed to amplify PCR products corresponding to five human glucose transporters (SGLT1, GLUT1, GLUT2, GLUT3 and GLUT4). RT-PCR was carried out to detect transcripts of glucose transporters present in pooled cDNA libraries from normal human articular cartilage. Glucose transporter expression levels were quantified relative to β-actin. Polyclonal antibodies raised against synthetic peptides corresponding to exofacial loops of glucose transporters identified in chondrocytes by PCR were used in flow cytometry studies and the results analysed by WinMDI software.

The results of RT-PCR studies confirmed that GLUT1 and GLUT3 are expressed in normal human articular cartilage and in immortalized chondrocytes. Purification, cloning and sequencing of the PCR product corresponding to GLUT1 confirmed expression of GLUT1 in adult human chondrocytes. The sequence obtained was 100 % identical to nucleotides 1059 to 1451 of the Homo sapiens facilitated glucose transporter, member 1, GLUT1 (SLC2A1). The partial sequence obtained from the human cartilage cDNA library was deposited in Genbank (accession number AY034633). Sequencing of the 457 base pair PCR product corresponding to GLUT3 confirmed presence of GLUT3 mRNA in chondrocytes. The sequence was 100 % identical to nucleotides 1144 to 1601 of the Homo sapiens facilitated glucose transporter, member 3, GLUT3 (SLC2A3). The sequence was deposited in Genbank (accession number AY034634). We found no evidence for the expression of GLUT2, GLUT4 or the sodium-dependent glucose transporter SGLT1 in human cartilage. When normalized to β-actin, expression of GLUT1 mRNA was approximately 2.5-fold higher than GLUT3. FACS analysis demonstrated that GLUT1 and GLUT3 were expressed in more than 90 % of monolayer chondrocytes examined (Fig. 1). In chondrocytes maintained in monolayer culture for up to 2 weeks, expression levels decreased (P < 0.005, Student’s t test) to 64.96 % (GLUT1) and 84.44 % (GLUT3) (means ± S.E.M., n = 3). GLUT expression levels in chondrocytes encapsulated in alginate beads were significantly (P < 0.0005, Student’s t test) reduced to 7.21 % (GLUT1) and 19.46 % (GLUT3) (means ± S.E.M., n = 3).

This study demonstrates, for the first time, that at least two facilitative glucose transporters are expressed by human articular chondrocytes. The presence of GLUT1 in cartilage was expected as this glucose transporter is ubiquitously expressed. Expression of GLUT3 in cartilage was surprising, as this transporter is normally found in regions of high metabolic activity in the central nervous system, the placenta, testes and spermatozoa (Haber et al. 1993). Although the expression of GLUT4 has been reported in developing mouse craniofacial mesenchyme, immature mouse cartilage (Vannucci et al. 2000) and in mouse growth plate chondrocytes (Wang et al. 1999), our studies suggest that in normal adult cartilage GLUT4 is not present and GLUTs 1 and 3 predominate. Given the central role of glucose in normal chondrocyte physiology and metabolism in situ, its regular provision is likely to have a profound influence on the metabolic activity and survival of chondrocytes in articular cartilage matrices. The flow cytometry studies indicate that GLUT1 and GLUT3 proteins are expressed in human chondrocytes but are downregulated in long-term alginate cultures.

Figure 1. FACS analysis of immortalized human chondrocytes in monolayer probed with polyclonal antibodies to GLUT1 and GLUT3 and secondary FITC-conjugated anti-rabbit IgG. A, scatter plot of chondrocytes based on cell size and granularity. B, fluorescence from control cells incubated with non-immune serum. C and D, specific fluorescence from cells probed with antibodies to GLUT1 and GLUT3.

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