THE METABOLISM OF 2-FLUORO-2-DEOXY-D-GLUCOSE IN HUMAN CHONDROCYTES AND ITS INCORPORATION INTO KERATAN SULFATE PROTEOGLYCANS

The incorporation of 2-fluoro-2-deoxy-D-[C-14] glucose in proteoglycans was investigated in a cell culture system, where human articular chondrocytes were cultured in high-cell-density thin-layer soft agarose. The proteoglycans were solubilized from the culture medium and the cell layer fraction by extracting with a guanidine hydrochloride buffer and purified by an ion-exchange-chromatography (DEAE-Sepharose CL-GB). With enzymic decomposition experiments concerning the glycosaminoglycan side-chains it could be shown that 65-69% were digestible by keratanase, whereas 21-29% of the C-14-labeled proteoglycans were digested with chondroitinase AC/ABC. The main constituent of the 2-fluoro-2-deoxy-D-[C-14]glucose-metabolites present in the glycosaminoglycan side chains of the proteoglycans was 2-fluoro-2-deoxy-D-[C-14]galactose. Therefore, 2-fluoro-2-deoxy-D-glucose was preferentially incorporated into keratan sulfate. We investigated the effect of non-radioactive 2-fluoro-2-deoxy-D-glucose on UDP-sugar and proteoglycan biosynthesis after incubation periods of 1-30 h. A high 2-fluoro-2-deoxy-D-glucose concentration in the culture medium did not influence the pool size of UDP-N-acetylhexosamines, but UDP-D-glucose, UDP-D-galactose, UDP-D-glucuronic acid, UDP-2-fluoro-2-deoxy-D-glucose, UDP-2-fluoro-2-deoxy-D-galactose and UDP-2-fluoro-2-deoxy-D-glucuronic acid accumulated in the chondrocytes time dependently. In a pulse/chase experiment the retarded synthesis of fluorinated UDP-sugars was proved. The half-lives (t(1/2)) for UDP-2-fluoro-2-deoxy-D-glucose and UDP-2-fluoro-2-deoxy-D-galactose were about 7.7 h and 13.3 h, respectively. UDP-2-fluoro-2-deoxy-D-glucuronic acid could be found with delay. The incubation with 2-fluoro-2-deoxy-D-glucose and [C-14]glucosamine resulted in a decreased radioactive labelling of chondroitin sulfate and keratan sulfate.