NMR-SPECTROSCOPY OF CULTURED ASTROCYTES - EFFECTS OF GLUTAMINE AND THE GLIOTOXIN FLUOROCITRATE

Glial synthesis of glutamine, citrate, and other carbon skeletons, as well as metabolic effects of the gliotoxin fluorocitrate, were studied in cultured astrocytes with C-13 and P-31 NMR spectroscopy. [2-C-13]A cetate and [1-C-13]glucose were used as labeled precursors. In some ex periments glutamine (2.5 mM) was added to the culture medium. Fluoroci trate (20 mu M) inhibited the tricarboxylic acid (TCA) cycle without a ffecting the level of ATP. The net export of glutamine was reduced sig nificantly, and that of citrate increased similarly, consistent with a n inhibition of aconitase. Fluorocitrate (100 mu M) inhibited TCA cycl e activity even more and (without addition of glutamine) caused a 40% reduction in the level of ATP. In the presence of 2.5 mM glutamine, 10 0 mu M fluorocitrate did not affect ATP levels, although glutamine syn thesis was nearly fully blocked. The consumption of the added glutamin e increased with increasing concentrations of fluorocitrate, whereas t he consumption of glucose decreased. This shows that glutamine fed int o the TCA cycle, substituting for glucose as an energy substrate. Thes e findings may explain how fluorocitrate selectively lowers the level of glutamine and inhibits glutamine formation in the brain in vivo, vi z., not by depleting glial cells of ATP, but by causing a rerouting of 2-oxoglutarate from glutamine synthesis into the TCA cycle during inh ibition of aconitase. Analysis of the C-13 labeling of the C-2 versus the C-4 positions in glutamine obtained with [2-C-13]acetate revealed that 57% of the TCA cycle intermediates were lost per turn of the cycl e. Glutamine and citrate were the main TCA cycle intermediates to be r eleased, but a large amount of lactate formed from TCA cycle intermedi ates was also released, showing that recycling of pyruvate takes place in astrocytes.