Differential metabolic effects of glucosamine and N-acetylglucosamine in human articular chondrocytes A.R. Shikhman, D.C. Brinson, J. Valbracht, M.K.

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Differential metabolic effects of glucosamine and N-acetylglucosamine in human articular chondrocytes A.R. Shikhman, D.C. Brinson, J. Valbracht, M.K. Lotz Osteoarthritis and Cartilage Volume 17, Issue 8, Pages 1022-1028 (August 2009) DOI: 10.1016/j.joca.2009.03.004 Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Facilitated transport of GlcN and GlcNAc in human articular chondrocytes and other cell types. A. Time course of [3H]GlcN and [3H]GlcNAc uptake in chondrocytes. Facilitated transport of GlcN and GlcNAc in human normal articular chondrocytes was measured by [3H]GlcN and [3H]GlcNAc uptake, respectively. Chondrocytes actively import GlcN in a time-dependent fashion. The import of GlcNAc is not statistically significant. Cell-type specificity of GlcN (B) and GlcNAc transport (C). Facilitated transport of GlcN and GlcNAc was measured by [3H]GlcN and [3H]GlcNAc uptake, respectively, after 30min incubation with the labeled sugars. The following cell types were used: HC – normal human chondrocytes; SFB – normal human synovial fibroblasts; SW1353 – human chondrosarcoma cells; 293 – human embryonic kidney; BJ – normal human foreskin fibroblasts. Osteoarthritis and Cartilage 2009 17, 1022-1028DOI: (10.1016/j.joca.2009.03.004) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 GlcN but not GlcNAc inhibits facilitated glucose transport. Normal human chondrocytes were treated with GlcN or GlcNAc for 4h followed by measurement of facilitated glucose transport ([3H]2DG uptake). [3H]2DG uptake in unstimulated chondrocytes was considered as 100%. *P<0.05. Osteoarthritis and Cartilage 2009 17, 1022-1028DOI: (10.1016/j.joca.2009.03.004) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 GlcN inhibits plasma membrane incorporation of GLUT1 and 6. Chondrocytes were stimulated with IL-1β (1ng/ml) with or without GlcN (10mM) for 24h. Detection of GLUT1, 3 and 6 proteins in the chondrocyte plasma membranes was performed using Western blotting. Unstimulated chondrocytes were used as control. Osteoarthritis and Cartilage 2009 17, 1022-1028DOI: (10.1016/j.joca.2009.03.004) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 GlcN depletes intracellular ATP. Chondrocytes incubated in DMEM containing 2% serum and 5mM Glc were treated with various concentrations of GlcN or GlcNAc for 4h followed by measurement of intracellular ATP. Concentration of ATP in untreated chondrocytes was 1.7+/−0.8μM per 5×106 cells, and it was considered as 100%. *P<0.05. Osteoarthritis and Cartilage 2009 17, 1022-1028DOI: (10.1016/j.joca.2009.03.004) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Different effects of GlcN and GlcNAc on sulfated glycosaminoglycan (SGAG) (A) and HA (B) synthesis. A. SGAG synthesis was measured by metabolic labeling of normal and OA human knee chondrocytes with [35S]SO4. Chondrocytes were incubated with either GlcN or GlcNAc in DMEM containing 5mM glucose and 2% serum for 24h followed by the metabolic labeling with [35S]SO4 in the presence of the aminosugars. [35S]SO4 incorporation in untreated chondrocytes was considered as 100%. B. Chondrocytes were treated with either GlcN or GlcNAc in DMEM containing 5mM glucose and 2% serum for 72h. Hyaluronan (HA) concentration in chondrocyte supernatants was measured using an assay with hyaluronan binding protein. HA concentration in untreated chondrocytes was considered as 100%. Osteoarthritis and Cartilage 2009 17, 1022-1028DOI: (10.1016/j.joca.2009.03.004) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 GlcNAc increases expression of HAS-2 in human articular chondrocytes. Normal human articular chondrocytes were stimulated with 10mM GlcNAc for 24h. Expression of HAS-2 mRNA was analyzed by RT-PCR. Osteoarthritis and Cartilage 2009 17, 1022-1028DOI: (10.1016/j.joca.2009.03.004) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions