Glutamine metabolism can allow cells to meet both the anaplerotic and NADPH demands of growth. Since the 1950s, it has been clear that tumors consume large amounts of glutamine. The rate of consumption is not explained by protein synthesis because it exceeds the need for essential amino acids by 10-fold [49
]. Later studies revealed rapid but partial glutamine oxidation and secretion of glutamine-derived carbon as lactate (), establishing glutamine as an energy source in tumor cells [50
]. ‘Glutaminolysis,’ the metabolism of glutamine to lactate, is considered a hallmark of tumor cell metabolism [15
The proximal reactions of glutaminolysis occur in the mitochondria. The first step is catalyzed by phosphate-dependent glutaminases, which deamidate glutamine to form glutamate and ammonia. Interconversion of glutamine and glutamate is typically bidirectional, with glutamine formation catalyzed by glutamine synthetase. In tumors, however, the forward (towards glutamate) reaction is favored by overexpression of glutaminases and/or suppression of glutamine synthetase [51
]. Thus deamidation is a control point for glutamine metabolism in tumor cells. In xenografts, glutaminase expression is temporally correlated with maximal growth rate, and suppression of glutaminase activity limits tumor growth [55
Little is known about how tumor cells regulate glutaminase expression. Mammals have two major glutaminase activities, K-type (low Km
for glutamine, inhibited by glutamate) and L-type (high Km
, glutamate resistant). The human K-type enzyme is encoded by the GLS
gene, which yields several mRNAs due to alternative polyadenylation and splicing [58
], and the L-type enzyme is encoded by GLS2
. In general, tumor cells have K-type activity, although most cell lines express transcripts from both genes [52
]. This suggests that tumors can modulate glutaminase kinetics through relative levels of GLS
gene products, resulting in the ability to optimize glutaminase activity despite local fluctuations in glutamine and glutamate concentrations.
In some cells, glutamine-derived α-ketoglutarate (α-KG) is the major source of OAA. A large glutamine-based anaplerotic flux was suggested in rat glioma cells studied with 13
C NMR spectroscopy, when adding unlabeled glutamine to cultures containing 13
C-glucose suppressed labeling in TCA cycle intermediates [60
]. More recently, glutamine deprivation from fibroblasts was shown to reduce cellular pools of TCA cycle intermediates [61
]. Finally, NMR spectroscopy in human glioblastoma cells cultured with 13
C-labeled glutamine showed conclusively that glutamine contributed the bulk of anaplerotic carbon to the TCA cycle [47••
]. The co-existence of robust glucose and glutamine metabolism in these cells resulted in production of citrate molecules containing two glucose-derived carbons (from acetyl-CoA) and four glutamine-derived carbons (from OAA). Further utilization of citrate allowed the glucose-derived carbons to be transferred to fatty acids. Thus, glutamine-based anaplerosis is required in the way some tumor cells use TCA cycle intermediates for growth ().
Export of glutamine-derived malate to the cytoplasm short-circuits the TCA cycle but delivers substrate to malic enzyme for NADPH production. Evidence suggests that this can be the major source of NADPH in tumor cells. In human glioblastoma cells, glutaminolysis was predicted to produce more than enough NADPH for fatty acid synthesis; the surplus could presumably be used for nucleotide biosynthesis and maintenance of the glutathione pool [47••
]. This was true even though these same cells used glutamine as the major source of anaplerotic carbon. Overall, more than half of the glutamine-derived carbon was secreted as lactate and alanine.
The high rate of lactate and alanine secretion per mole of glutamine, similar to the apparent ‘wastefulness’ of the Warburg effect, has been observed in other proliferating cells [7
]. In contrast to the common perception that cells use glutamine primarily as a nitrogen source, glutamine metabolism in cancer cells results in excess intracellular nitrogen that must be secreted as alanine or ammonia. Glutaminase removes glutamine's amido group as ammonia. Surprisingly, in glioblastoma cells, the majority of glutamine's amino groups were also lost in α-KG-generating reactions (glutamate dehydrogenase and alanine aminotransferase) [47••
]. Therefore, utilization of glutamine as an anaplerotic precursor and source of NADPH results in the secretion of a large fraction of glutamine-derived carbon and nitrogen. Some of the secreted molecules (lactate, alanine) may subsequently be used as precursors for hepatic gluconeogenesis, ultimately providing more fuel for tumor metabolism. At first glance, these appear to be symptoms of metabolic inefficiency, but they may actually reflect a logical and specialized form of metabolism that enables cell growth and proliferation.