Tumor cell proliferation requires rapid synthesis of macromolecules including lipids, proteins, and nucleotides. Many tumor cells exhibit rapid glucose consumption, with most of the glucose-derived carbon being secreted as lactate despite abundant oxygen availability (the Warburg effect). Here, we used 13 C NMR spectroscopy to examine the metabolism of glioblastoma cells exhibiting aerobic glycolysis. In these cells, the tricarboxylic acid (TCA) cycle was active but was characterized by an efflux of substrates for use in biosynthetic pathways, particularly fatty acid synthesis. The success of this synthetic activity depends on activation of pathways to generate reductive power (NADPH) and to restore oxaloacetate for continued TCA cycle function (anaplerosis). Surprisingly, both these needs were met by a high rate of glutamine metabolism. First, conversion of glutamine to lactate (glutaminolysis) was rapid enough to produce sufficient NADPH to support fatty acid synthesis. Second, despite substantial mitochondrial pyruvate metabolism, pyruvate carboxylation was suppressed, and anaplerotic oxaloacetate was derived from glutamine. Glutamine catabolism was accompanied by secretion of alanine and ammonia, such that most of the amino groups from glutamine were lost from the cell rather than incorporated into other molecules. These data demonstrate that transformed cells exhibit a high rate of glutamine consumption that cannot be explained by the nitrogen demand imposed by nucleotide synthesis or maintenance of nonessential amino acid pools. Rather, glutamine metabolism provides a carbon source that facilitates the cell's ability to use glucose-derived carbon and TCA cycle intermediates as biosynthetic precursors.cancer ͉ glioblastoma ͉ Warburg effect ͉ glutaminolysis ͉ anaplerosis I n mammals, cell proliferation is controlled by signal transduction pathways stimulated by lineage-specific growth factors or, in tumors, constitutive activation of these pathways through oncogenic mutations. A proximal effect of signaling pathways is a robust increase in nutrient uptake (1-3). Cells must also allocate these molecules into appropriate metabolic pathways to produce and maintain pools of intermediates needed to synthesize macromolecules. Therefore, a complete understanding of the biology of cell proliferation will require a comprehensive understanding of the regulation of metabolic fluxes.Glucose and glutamine are two of the most abundant nutrients in plasma, and together they account for most carbon and nitrogen metabolism in mammalian cells. Rapid cell proliferation has been associated with a robust but apparently wasteful metabolism of glucose. In the 1920s, Otto Warburg demonstrated that ascites tumor cells had high rates of glucose consumption and lactate production despite availability of sufficient oxygen to oxidize glucose completely (4). The ''Warburg effect'' is taken to be a metabolic hallmark of aggressive tumors; however, the phenotype is also observed in nontransformed cells during rapid proliferation (2, 5). Gl...

Mammalian cells fuel their growth and proliferation through the catabolism of two main substrates: glucose and glutamine. Most of the remaining metabolites taken up by proliferating cells are not catabolized, but instead are used as building blocks during anabolic macromolecular synthesis. Investigations of phosphoinositol 3-kinase (PI3K) and its downstream effector AKT have confirmed that these oncogenes play a direct role in stimulating glucose uptake and metabolism, rendering the transformed cell addicted to glucose for the maintenance of survival. In contrast, less is known about the regulation of glutamine uptake and metabolism. Here, we report that the transcriptional regulatory properties of the oncogene Myc coordinate the expression of genes necessary for cells to engage in glutamine catabolism that exceeds the cellular requirement for protein and nucleotide biosynthesis. A consequence of this Myc-dependent glutaminolysis is the reprogramming of mitochondrial metabolism to depend on glutamine catabolism to sustain cellular viability and TCA cycle anapleurosis. The ability of Myc-expressing cells to engage in glutaminolysis does not depend on concomitant activation of PI3K or AKT. The stimulation of mitochondrial glutamine metabolism resulted in reduced glucose carbon entering the TCA cycle and a decreased contribution of glucose to the mitochondrial-dependent synthesis of phospholipids. These data suggest that oncogenic levels of Myc induce a transcriptional program that promotes glutaminolysis and triggers cellular addiction to glutamine as a bioenergetic substrate.cancer ͉ mitochondria

Glutamate dehydrogenase (GDH) is regulated by both positive (leucine and ADP) and negative (GTP and ATP) allosteric factors. We hypothesized that the phosphate potential of ␤-cells regulates the sensitivity of leucine stimulation. These predictions were tested by measuring leucine-stimulated insulin secretion in perifused rat islets following glucose depletion and by tracing the nitrogen flux of [2-15 N]glutamine using stable isotope techniques. The sensitivity of leucine stimulation was enhanced by long time (120-min) energy depletion and inhibited by glucose pretreatment. After limited 50-min glucose depletion, leucine, not ␣-ketoisocaproate, failed to stimulate insulin release. ␤-Cells sensitivity to leucine is therefore proposed to be a function of GDH activation. Leucine increased the flux through GDH 3-fold compared with controls while causing insulin release. High glucose inhibited flux through both glutaminase and GDH, and leucine was unable to override this inhibition. These results clearly show that leucine induced the secretion of insulin by augmenting glutaminolysis through activating glutaminase and GDH. Glucose regulates ␤-cell sensitivity to leucine by elevating the ratio of ATP and GTP to ADP and P i and thereby decreasing the flux through GDH and glutaminase. These mechanisms provide an explanation for hypoglycemia caused by mutations of GDH in children.In addition to glucose, amino acids and other metabolic fuels are important stimulants of insulin secretion from pancreatic ␤-cells. Leucine, which has been studied intensively, may stimulate insulin release through two different mechanisms. The first involves transamination of leucine to ␣-ketoisocaproate (KIC) 1 and subsequent mitochondrial oxidation. The second promotes insulin release via allosteric activation of glutamate dehydrogenase (GDH) causing oxidation of glutamate to the Krebs cycle intermediate, ␣-ketoglutarate, plus ammonia. The importance of the latter mechanism has been highlighted recently by the discovery of a dominant form of congenital hyperinsulinism associated with mutations of GDH leading to a gain of enzyme activity, because sensitivity to inhibition by GTP and ATP is impaired (1-3). Affected children have increased ␤-cell responsiveness to leucine and are susceptible to acute hypoglycemia following a high protein meal (4). The involvement of GDH may explain the observation that, in contrast to other amino acids, leucine-stimulated insulin secretion (LSIS) is suppressed by high glucose. For example, Gao et al. (5) reported that glucose inhibits leucine stimulation of glutaminolysis and insulin secretion in isolated mouse islets, presumably by increasing intracellular ATP and GTP while decreasing ADP and thus inhibiting GDH activity.GDH has also been proposed by Maechler and Wollheim (6) to play an essential role in glucose-mediated insulin secretion by acting in the reverse direction to catalyze production of glutamate, which is hypothesized to work as a cofactor in the process leading to exocytosis of insulin granules. T...

Children with hypoglycemia due to recessive loss of function mutations of the ␤-cell ATP-sensitive potassium (K ATP ) channel can develop hypoglycemia in response to protein feeding. We hypothesized that amino acids might stimulate insulin secretion by unknown mechanisms, because the K ATP channel-dependent pathway of insulin secretion is defective. We therefore investigated the effects of amino acids on insulin secretion and intracellular calcium in islets from normal and sulfonylurea receptor 1 knockout (SUR1؊/؊) mice. Even though SUR1؊/؊ mice are euglycemic, their islets are considered a suitable model for studies of the human genetic defect. SUR1؊/؊ islets, but not normal islets, released insulin in response to an amino acid mixture ramp. This response to amino acids was decreased by 60% when glutamine was omitted. Insulin release by SUR1؊/؊ islets was also stimulated by a ramp of glutamine alone. Glutamine was more potent than leucine or dimethyl glutamate. Basal intracellular calcium was elevated in SUR1؊/؊ islets and was increased further by glutamine. In normal islets, methionine sulfoximine, a glutamine synthetase inhibitor, suppressed insulin release in response to a glucose ramp. This inhibition was reversed by glutamine or by 6-diazo-5-oxo-L-norleucine, a non-metabolizable glutamine analogue. High glucose doubled glutamine levels of islets. Methionine sulfoximine inhibition of glucose stimulated insulin secretion was associated with accumulation of glutamate and aspartate. We hypothesize that glutamine plays a critical role as a signaling molecule in amino acid-and glucosestimulated insulin secretion, and that ␤-cell depolarization and subsequent intracellular calcium elevation are required for this glutamine effect to occur.