ISOLATION OF CARBOHYDRATE METABOLIC CLONES FROM CULTURED ASTROCYTES

Astrocytes are the principal sites of glycogen synthesis in the nervou s tissue. Growing evidence shows that there are many types of astrocyt es. The aim of the present investigation was to isolate different type s of astrocytes that display different carbohydrate anabolism. Astrocy tes from newborn rat brain were directly cloned from primary cultures without a previous transformation. Many clones were obtained, and they were termed CP clones. Another series of clones, termed SV clones, we re obtained after the transfection of the primary cultures by the SV40 T antigen. The effectiveness of the transfection was verified by the rate of DNA synthesis using flow cytometry and by the presence of plas mid DNA in the genomic DNA of the astrocytes using the Southern blot m ethod. After the transfection, the growth velocity increased greatly, The size and shape of the astrocytes were the same for each cell in a given clone, regardless of the cloning method utilized. However, these sizes and shapes could be different from one clone to another in CP c lones, whereas all the astrocytes of all the SV clones looked like eac h other, All the clones obtained stained positively with anti-glial fi brillary acidic protein antibodies. Glycogen stained in the clones usi ng concanavalin A-horseradish peroxidase. The glycogen content was als o measured using biochemical analysis. Concordant results obtained usi ng these two methods showed that some clones contained an important qu antity of glycogen while other clones contained a small amount;, in th e CP series as well as in the SV series. This property was the same fo r the intracellular glucose concentrations. The activity of the glucon eogenic enzyme fructose-1,6-bisphosphatase was measured in each clone using spectrophotometry. This activity was also significantly differen t from one clone to another. The clones containing large amounts of gl ycogen had important fructose-1,6-bisphosphatase activity. The present results show that it is possible to clone astrocytes either directly from primary cultures without immortalization or after their transform ation. when analyzing these clones, it appears that carbohydrate anabo lism can be significantly different from one astrocyte to another. Thi s difference may also exist in vivo. (C) 1996 Wiley-Liss, Inc.