Introduction: Glutamine is known to play a central role in cancer cell metabolism. It serves as a major source of nitrogen for amino acids used in protein synthesis and purines and pyrimidines used in nucleic acid synthesis. The carbon backbone generally serves as an anaplerotic source for the TCA cycle. Removal of both amine groups produces alpha alpha-ketoglutarate and subsequently succinate to resupply the four-carbon units depleted for biosynthetic purposes, such as amino acids. It may also play an anaplerotic role during fatty acid synthesis, if oxaloacetate produced by ATP citrate lyase (ACL) in the cytoplasm is converted to pyruvate.

Our lab is examining ACL as a possible target for cancer therapy. Preliminary studies have shown that inhibition of this enzyme reduces cell proliferation, presumably because fatty acids needed for daughter cell phospholipids cannot be produced in sufficient quantities (1). To understand the metabolic consequences of such inhibition, we have been exploring the use of ¹³C NMR spectroscopy to non-invasively monitor metabolism in real time. In this work, we describe results obtained recently with human glioma cells perfused with medium containing [3-¹³C]glutamine] alone and in combination with [1,6-¹³C₂]glucose.

Methods: SF188 cells (human glioma grade 4, Brain Tumor Research Center, UCSF) were grown with DMEM (supplemented with 10% serum and 50 µg/ml gentamicin) in porous collagen microcarriers (Hyclone, Logan, UT). The microcarriers were packed densely inside a 20-mm NMR tube (2) and perfused continuously (37 °C, pH = 7.2, dissolved oxygen = 0.2 mM) inside a 9.4T spectrometer (Varian, Palo Alto, CA) (3). ³¹P spectral parameters were: 60° pulse width, 1000 ms repetition time, 4096 points, and 15000 Hz spectral width. NTP levels indicated that the number of viable cells in the NMR tube was approximately 10⁹ (4). ¹³C spectra were acquired with 60° pulses, a repetition time of 1200 ms, 4096 points, 25000 Hz spectral width and ¹H bi-level WALTZ-16 decoupling. Each spectrum was aquired with 750 scans in 15-min. Cells were initially fed DMEM with un-enriched glucose and glutamine while background spectra were acquired. Subsequently, the un-enriched medium was completely replaced with DMEM containing either 4 mM [3-¹³C]glutamine (Sigma-Aldrich, St. Louis, MO) or 10 mM [1,6-¹³C₂] glucose (Cambridge Isotopes, Andover, MA).

Results and Discussion: Following addition of [3-¹³C]glutamine, [3-¹³C]glutamate was immediately detected and label in the glutamate pool reached saturation within 1.5 hours. Label at C-2 of glutamate, which requires complete TCA cycle activity (3), was not observed until 30 minutes after the addition of the labeled glutamine. It reached saturation approximately 2.5 hours later. Labeling in C-2 and C-3 of aspartate was detected within 15 minutes of the start of the addition of [3-¹³C]glutamine. The amount of label present at both carbons was comparable and both saturated within approximately 1 hour. Labeling in asparate is believed to reflect labeling in oxaloacetate since the two compounds are in rapid equilibrium. The equivalent labeling at the two central carbons supports the belief that when label from C-3 of glutamate is transferred to C-3 of alpha-keto glutarate, it is subsequently distributed to both C-2 and C-3 and of succinate, since succinate is a symmetrical molecule. Label was also detected in C-2 and C-3 of malate, within 45 minutes of the addition of the labeled glutamine. To our knowledge, this is the first report of real-time detection of an actual TCA cycle intermediate in cultured cells with 13C NMR spectroscopy.

A small amount of label was detected in C-2 and C-3 of lactate, which demonstrates that four-carbon units from the TCA cycle are converted to pyruvate by either malic enzyme, the pyruvate-citrate cycle associated with lipid synthesis or other similar pathways. Some labeled pyruvate apparently also re-entered the TCA cycle to form citrate since labeled fatty acyl groups were detected in significant quantities. Cells perfused with [3-¹³C]glutamine and [1,6-¹³C₂]glucose simultaneously produced large amounts of [3,4-¹³C₂]glutamate. This was very likely produced from [3,4-¹³C₂]citrate, that was formed when [2-¹³C]acetate combined with [2-¹³C]oxaloacetate by citrate synthase. This result alone strongly supports that belief that glutamine plays a major anaplerotic role in SF188 cells.

We are currently developing a mathematical model to determine pathway fluxes from our ¹³C kinetic data. We expect this model to become a powerful tool to facilitate the development of new therapeutic approaches for the treatment of cancer.

Conclusions: Real-time ¹³C NMR spectroscopy can provide unique information about metabolic pathways of cultured cells that would be difficult to obtain with any other non-invasive method. Future studies will examine the effects of ACL inhibition (by both pharmacological agents and RNA interference techniques) on fatty acyl synthesis, anaplerosis and TCA cycle activity.

Acknowledgements This work was supported by the National Cancer Institute and the Abramson Family Cancer Research Institute of the University of Pennsylvania.

References: (1) Hatzivassiliou G, Zhao F, Bauer DE, Andreadis C, Shaw AN, Dhanak D, Hingorani SR, Tuveson DA, Thompson CB. ATP citrate lyase inhibition can suppress tumor cell growth. Cancer Cell. 2005 8(4):311-21.

(2) Mancuso A, Zhu A, Beardsley NJ, Glickson JD, Wehrli S, Pickup S. Artificial tumor model suitable for monitoring 31P and 13C NMR spectroscopic changes during chemotherapy-induced apoptosis in human glioma cells. Magn Reson Med. 2005 54(1):67-78.

(3) Mancuso A, Beardsley NJ, Wehrli S, Pickup S, Matschinsky FM, Glickson JD. Real-time detection of 13C NMR labeling kinetics in perfused EMT6 mouse mammary tumor cells and betaHC9 mouse insulinomas. Biotechnol Bioeng. 2004 30;87(7):835-48.

(4) Mancuso A, Fernandez EJ, Blanch HW, Clark DS. A nuclear magnetic resonance technique for determining hybridoma cell concentration in hollow fiber bioreactors. Bio/Technology (Nature Publishing). 1990 8(12):1282-5.