Role of Glucose and Glutamine Metabolism in the Growth of a Human T -lymphoblastoid Line, Jurkat
Gayton, Marshall George
Glacken, W. Michael, Sc. D.
Master of Science
The interrelationship between glucose and glutamine metabolism was examined using Jurkat, a human T-lymphoblastoid line. This study utilized three experimental approaches to examine the metabolism of Jurkat: (i) an in vitro whole cell 'plate assay' , (ii) continuous culture bioreactor pulse changes, (iii) a proliferative assay, using the tetrazolium salt MTT. (i) In the plate assay, cells were incubated in Kerbs-Ringer buffer at an initial density of 2-7x 1 ()6 cells/mi. The utilization of select nutrients, and subsequent product formation was · followed over a period of 8 hours. Both intercellular and extracellular nutrient levels are measured in this technique. (ii) Jurkat maintained at a dilution rate of 0.372 day-I was subjected to a number of pulse changes( tO mM glucose, 5 mM pyruvate, 4 mM glutamine, 10 mM oxaloacetate plus 2.5 m M glutamate, and 0.5x amino acids) and the resulting shifts in metabolism were followed The 10 mM oxaloacetate and 2.5 mM glutamate pulse resulted in a marked decrease glutamine utilization and a subsequent drop in ammonium production. This pulse also provided the only instance in which increased levels of aspartic acid were detectable. (iii) The MTT assay was modified to replace the more commonly used 'growth assay' for the measurement of cellular proliferation/activation. Results observed in this work indicate:(a) that both aspartate-aminotransferase and alanine-aminotranferase are involved in the breakdown of glutamine;(b) aspartate's carbon skeleton is derived from glutamine, while alanine's carbon skeleton comes from glucose;(c) while glutamine utilization does not alter glucose metabolism in Jurkat, the presence of glucose not only reduced the rate of glutamine utilization, it also altered the products formed from glutamine, (d) the inability to regenerate cytosolic NAD via mitochondrial transport may contribute to the production of high levels of lactate from glucose.