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More recognition that ketosis can restrict growth of cancer cells.

Amino Acid and Glucose Fermentation Maintain ATP Content in Mouse and Human Malignant Glioma Cells

Talking Cancer With Professor Thomas Seyfried: New Study Confirms that Cancer Cells Ferment Glutamine

Abstract

Energy is necessary for tumor cell viability and growth. Aerobic glucose-driven lactic acid fermentation is a common metabolic phenotype seen in most cancers including malignant gliomas. This metabolic phenotype is linked to abnormalities in mitochondrial structure and function. A luciferin-luciferase bioluminescence ATP assay was used to measure the influence of amino acids, glucose, and oxygen on ATP content and viability in mouse (VM-M3 and CT-2A) and human (U-87MG) glioma cells that differed in cell biology, genetic background, and species origin. Oxygen consumption was measured using the Resipher system. Extracellular lactate and succinate were measured as end products of the glycolysis and glutaminolysis pathways, respectively. The results showed that: (1) glutamine was a source of ATP content irrespective of oxygen. No other amino acid could replace glutamine in sustaining ATP content and viability; (2) ATP content persisted in the absence of glucose and under hypoxia, ruling out substantial contribution through either glycolysis or oxidative phosphorylation (OxPhos) under these conditions; (3) Mitochondrial complex IV inhibition showed that oxygen consumption was not an accurate measure for ATP production through OxPhos. The glutaminase inhibitor, 6-diazo-5-oxo-L-norleucine (DON), reduced ATP content and succinate export in cells grown in glutamine. The data suggests that mitochondrial substrate level phosphorylation in the glutamine-driven glutaminolysis pathway contributes to ATP content in these glioma cells. A new model is presented highlighting the synergistic interaction between the high-throughput glycolysis and glutaminolysis pathways that drive malignant glioma growth and maintain ATP content through the aerobic fermentation of both glucose and glutamine.

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Conclusions

Our data show that glutamine could maintain sufficient ATP content in either the presence or absence of oxygen through mitochondrial substrate-level phosphorylation in the glutaminolysis pathway during glucose deprivation. No other amino acid had a stimulatory effect on ATP content comparable to that of glutamine in the mouse and human malignant glioma cells that we evaluated. The data show that oxygen consumption and lactic acid production are not accurate quantitative measures of ATP content through OxPhos or glycolysis, respectively, in these glioma cell lines. We also found that ATP content through OxPhos alone was unable to maintain bioluminescence of glioma cells in the absence of glucose and glutamine, indicating that these metabolites are both necessary and sufficient for maintaining growth of these glioma cells. A new metabolic model is presented highlighting the synergistic interaction between the high-throughput glycolysis and glutaminolysis pathways that converge on anabolic intermediates and maintain ATP content through the aerobic fermentation of both glucose and glutamine. Finally, the findings from this study can provide a general mechanism explaining the therapeutic efficacy seen from in vivo studies where the simultaneous restriction of glycolysis and glutaminolysis under nutritional ketosis enhance survival of young and adult mice bearing malignant brain tumors (Mukherjee et al., Citation2019; Citation2023; Seyfried et al., Citation2021; Citation2022)

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