Differential metabolic effects of glucosamine and N-acetylglucosamine in human articular chondrocytes

Abstract

Objective: Aminosugars are commonly used to treat osteoarthritis; however, molecular mechanisms mediating their anti-arthritic activities are still poorly understood. This study analyzes facilitated transport and metabolic effects of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) in human articular chondrocytes.

Methods: Human articular chondrocytes were isolated from knee cartilage. Facilitated transport of glucose, GlcN and GlcNAc was measured by uptake of [3H]2-deoxyglucose, [3H]GlcN and [3H]GlcNAc. Glucose transporter (GLUT) expression was analyzed by Western blotting. Production of sulfated glycosaminoglycans (SGAG) was measured using [(35)S]SO4. Hyaluronan was quantified using hyaluronan binding protein.

Results: Chondrocytes actively import and metabolize GlcN but not GlcNAc and this represents a cell-type specific phenomenon. Similar to facilitated glucose transport, GlcN transport in chondrocytes is accelerated by cytokines and growth factors. GlcN non-competitively inhibits basal glucose transport, which in part depends on GlcN-mediated depletion of ATP stores. In IL-1beta-stimulated chondrocytes, GlcN inhibits membrane translocation of GLUT1 and 6, but does not affect the expression of GLUT3. In contrast to GlcN, GlcNAc accelerates facilitated glucose transport. In parallel with the opposing actions of these aminosugars on glucose transport, GlcN inhibits hyaluronan and SGAG synthesis while GlcNAc stimulates hyaluronan synthesis. GlcNAc-accelerated hyaluronan synthesis is associated with upregulation of hyaluronan synthase-2.

Conclusion: Differences in GlcN and GlcNAc uptake, and their subsequent effects on glucose transport, GLUT expression and SGAG and hyaluronan synthesis, indicate that these two aminosugars have distinct molecular mechanisms mediating their differential biological activities in chondrocytes.

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 30 min incubation with the labeled sugars. The following cell types were used: HC – normal human chondrocytes; SFB – normal human synovial fibroblasts; SW1353 – human chondrosarcoma cell line; 293 – human embryonic kidney; BJ – normal human foreskin fibroblasts;

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 30 min incubation with the labeled sugars. The following cell types were used: HC – normal human chondrocytes; SFB – normal human synovial fibroblasts; SW1353 – human chondrosarcoma cell line; 293 – human embryonic kidney; BJ – normal human foreskin fibroblasts;

Fig. 2. GlcN but not GlcNAc inhibits facilitated glucose transport

Normal human chondrocytes were treated with GlcN or GlcNAc for 4 hours followed by measurement of facilitated glucose transport ([³H]2DG uptake). [³H]2DG uptake in unstimulated chondrocytes was considered as 100%. *p<0.05

Fig. 3. GlcN inhibits plasma membrane incorporation of GLUT1 and 6

Chondrocytes were stimulated with IL-1β (1ng/ml) with or without GlcN (10 mM) for 24 h. Detection of GLUT1, 3 and 6 proteins in the chondrocyte plasma membranes was performed using Western blotting. Unstimulated chondrocytes were used as control.

Fig. 4. GlcN depletes intracellular ATP

Chondrocytes incubated in DMEM containing 2% serum and 5 mM Glc were treated with various concentrations of GlcN or GlcNAc for 4 hours followed by measurement of intracellular ATP. Concentration of ATP in untreated chondrocytes was 1.7+/−0.8 μM per 5 × 10⁶ cells, and it was considered as 100%. *p<0.05

Fig. 5. Different effects of GlcN and GlcNAc on sulfated glycosaminoglycan (SGAG)(A) and hyaluronic acid (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 5 mM glucose and 2% serum for 24 h 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 5 mM glucose and 2% serum for 72 h. Hyaluronan (HA) concentration in chondrocyte supernatants was measured using an assay with hyaluronan binding protein. HA concentration in untreated chondrocytes was considered as 100%.

Fig. 6. GlcNAc increases expression of hyaluronan synthase-2 (HAS-2) in human articular chondrocytes

Normal Human articular chondrocytes were stimulated with 10 mM GlcNAc for 24 hours. Expression of HAS-2 mRNA was analyzed by RT-PCR.