L-PROLINE AS AN ALTERNATIVE ENERGY SUBSTRATE FOR GLUCOSE-DEPRIVED ASTROCYTES

The catabolism of L-proline in mammalian cells is mediated by mitochondrial enzymes that can oxidize proline to provide energy for mitochondrial ATP regeneration. To investigate the potential of astrocytes to consume and metabolize L-proline, we incubated cultured primary rat astrocytes with L-proline in the absence or the presence of other energy substrates and investigated L-proline consumption, cellular ATP content and cell viability. In the absence of glucose, the cells consumed L-proline which allowed them to maintain a high cellular ATP level as long as extracellular L-proline was detectable. This L-proline consumption was saturable and followed apparent Michaelis-Menten kinetics with a calculated K_M value of 320 µM and a V_max value of 100 nmol/(h x mg). The potential of 1 mM L-proline to maintain a high cellular ATP content in starved astrocytes and to prevent cell death was almost identical to that found for 1 mM glucose and co-application of both substrates had additive ATP-maintaining effects. The presence of glucose, glucose-derived lactate or other energy substrates severely slowed down astrocytic L-proline consumption. L-Proline consumption was lowered in a concentration-dependent manner by known inhibitors of proline dehydrogenase. Application of L-proline prevented the rapid loss in cellular ATP level and the subsequent toxicity induced by inhibitors of the mitochondrial uptake of pyruvate and fatty acids. These protective effects of proline were abolished by an inhibitor of proline dehydrogenase. The data presented demonstrate that L-proline is an excellent energy substrate for cultured astrocytes, especially for conditions of limited availability of other energy substrates.