Effects of glucosamine sulfate on intracellular UDP-hexosamine and UDP-glucuronic acid levels in bovine primary chondrocytes 1

SummaryObjectiveTo analyze the effects of exogenously added glucose (Glc), glucosamine (GlcN) and glucosamine sulfate (GS) on the intracellular UDP-hexoses (UDP-Hex), UDP-N-acetylhexosamines (UDP-HexN) and UDP-glucuronic acid (UDP-GlcA) levels in bovine primary chondrocytes.MethodsChondrocytes were incubated with different concentrations of Glc, GlcN and GS either in high- or low-glucose DMEM for up to 120 min to analyze the intracellular levels of UDP-Hex, UDP-GlcA and UDP-HexN by a reversed-phase high-performance liquid chromatography–electrospray ionization mass spectrometry analysis. Glycosaminoglycan (GAG) synthesis rate and aggrecan mRNA expression levels were quantified using 35S-sulfate incorporation assay and quantitative real-time RT-PCR, respectively. The cells were cultivated for 2 days or 8 days before UDP-sugar analysis.ResultsLevels of UDP-HexN and UDP-GlcA were unchanged at 10 μM concentration of GS in low-glucose DMEM, while addition of 1 mM GlcN or GS in low-glucose DMEM for 10 min increased UDP-HexN level. The highest intracellular level of UDP-HexN was reached at 30 min after addition of 1 mM GS to the cells. The intracellular contents of UDP-HexN and UDP-GlcA related to UDP-Hex were higher after prolonged cultivation of chondrocytes for 8 days compared with 2-day-old cultures. Aggrecan mRNA expression and GAG synthesis remained at control level after the cells were treated with 10, 100 μM or 1 mM of GS for 24 h.ConclusionPhysiologically relevant level of GS could not increase the intracellular UDP-HexN and UDP-GlcA levels in bovine primary chondrocyte, while longer-time culture itself appeared to increase the intracellular UDP-HexN and UDP-GlcA levels.