**1. Introduction**

28 Dehydrogenases

4):761-768.

*Ther S* Vol.1(No.:2.

evolution. *Cell* Vol.100(No. 1):157-168.

of nucleophile activation. *Proteins* Vol.57(No. 4):758-771.

family. *Eur J Biochem* Vol.251(No. 3):549-557.

Wang J, Sakariassen PO, Tsinkalovsky O*, et al.* (2008) CD133 negative glioma cells form tumors in nude rats and give rise to CD133 positive cells. *Int J Cancer* Vol.122(No.

Weiner D, Levy Y, Khankin EV & Reznick AZ (2008) Inhibition of salivary amylase activity by cigarette smoke aldehydes. *J Physiol Pharmacol* Vol.59 Suppl 6(No.:727-737. Weissman IL (2000) Stem cells: units of development, units of regeneration, and units in

White H, Smith L, Gentry T & Balber A (2011) Mechanisms of Action of Human Aldehyde Dehydrogenase Bright Cells in Therapy of Cardiovascular Diseases: Expression Analysis of Angiogenic Factors and Aldehyde Dehydrogenase Isozymes. *J Stem Cell Res* 

Wymore T, Hempel J, Cho SS, Mackerell AD, Jr., Nicholas HB, Jr. & Deerfield DW, 2nd (2004) Molecular recognition of aldehydes by aldehyde dehydrogenase and mechanism

Yoshida A, Rzhetsky A, Hsu LC & Chang C (1998) Human aldehyde dehydrogenase gene

Glutamate Dehydrogenase (GDH) catalyzes the oxidative conversion of glutamate to alpha ketoglutarate and ammonium supplying the TCA cycle with intermediates in support of anaplerosis (**Figure 1 Rxn1**). Conversely GDH catalyzes the reductive amination of alpha ketoglutarate and ammonium producing glutamate when the TCA cycle pool is filled. The net GDH flux resulting from these bidirectional fluxes can be obtained from the conversion of either 15N labeled glutamate and analyzing 15N ammonium or from 15N labeled NH4+ and monitoring 15N labeled glutamate. Besides deamination and 15N NH4+ production*,* 15N labeled glutamate can be converted to 15N labeled amino acids, most prominently alanine, via transamination reactions (**Figure 1, Rxn2**). In contrast to glutamate deamination which yields net keto acid production for anaplerosis [1], transamination does not yield net keto acid production (consuming a keto acid e.g. pyruvate in the process of generating alpha ketoglutarate). Under physiological conditions plasma glutamate concentration, 10-20uM, is limiting for GDH flux supplying TCA intermediates while plasma glutamine concentration, 600uM, is not [2]. The conversion of 15N amide labeled glutamine to 15N ammonium (**Figure1, Rxn1**) approximates the net glutaminase flux generating glutamate and ammonium, both potential substrates for GDH. Indeed GDH can also incorporate the amide derived 15N ammonium and alpha ketoglutarate into glutamate (**Figure 1, Rxn3**, reductive amination) which can subsequently transaminate with pyruvate generating 15N alanine [3]. Noteworthy this glutaminolytic anabolic pathway providing glutamate has been proposed as the primary metabolic transformation in tumor cells [4]. **Figure 1, Rxn 3** also illustrates how ammonium production from the 15N amide of glutamine may underestimate the true glutaminase flux; to the extent that this occurs, it contributes to differences in estimated net glutaminase fluxes between the chemically measured glutamine disappearance and 15N amide ammonium appearance. Glutamine labeled with 15N in the amino position provides

© 2012 Friday et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

an assessment of net GDH flux (**Figure 1,Rxn1)** as 15N NH4+ and, or, ALT flux as 15N alanine produced(**Figure1,Rxn2**).

Role of Glutamate Dehydrogenase in Cancer Growth and Homeostasis 31

addition, in vivo administered [2-(14)C] labeled glutamate was taken up by tumors with 14C distributed more into protein and lactate than in normal tissues [ 8 ]. In line with this early finding, a recent study[9] showed[U-(13)C] glucose contributed less than 50% of the acetyl COA pool in human brain tumors consistent with glutaminolysis and GDH's role in

**3. Intracellular glutamate and alpha ketoglutarate are in near equilibrium** 

In most cells intracellular glutamate and alpha ketoglutarate are in near equilibrium[4,10], and changes in TCA cycle intermediates(αKG) as well as the redox state(NADPH/NADP), energy charge(ADP,GTP) and cell pH shift the GDH catalyzed flux to net production or consumption of αKG (**Figure 2**). Normally pyruvate (glucose) provides the TCA cycle with pool intermediates while generated glutamate is transaminated (NH4+/GLN ratio<1, **Figure 1 Rxn3**). In cancer cells, glucose is shunted into aerobic glycolysis (Warburg effect, [11]) and the TCA cycle intermediates are reduced as the result of cataplerosis as evidenced by lower intracellular glutamate [7]. This reduction in TCA cycle intermediates "pulls" glutamate through GDH generating αKG as evidenced by the higher steady state NH4+/GLN ratio>1, **Figure 1, Rxn1** and consistent with glutamate (glutaminolysis) supporting anaplerosis (**Figure 2**). As a corollary, the ammonium to alanine produced ratio increases [7] reflecting the increased GDH and decreased ALT flux as the result of reduced intramitochondrial pyruvate(metabolized in cytosol to lactate, **Figure 2**). Thus the increased glutamate flux through GDH generates αKG while sparing keto acid consumption (reduced

**4. Glutamate is generated by extra- and intracellular glutaminases** 

Glutaminolysis as illustrated in **Figure 2** is associated with the increased expression of both the extrinsic cell membrane phosphate independent glutaminase/gamma glutamyltransferase/gamma glutamyltranspeptidase (PIG, GGT, GGTP) which generates extracellular glutamate [2,12] and intracellular phosphate dependent glutaminases, Phosphate dependent glutaminases (PDG,GLS1 and GAC, [13,14]) which generates glutamate cytosolically [2,13]; extracellular glutamate can be transported(GLAST, **Figure2**) into the cytosol functioning as an inhibitor of the intracellular glutaminases[2]. Noteworthy, c-myc signaling up-regulates both the cell membrane glutamine transporter (ASC, **Figure 2**) and the intracellular glutaminases in cancer cells [15]. On the other hand, increased expression of the extracellular PIG is also a hallmark of cancer cells [16] and PIG hydrolysis of ϒ-glutamyl-tagged fluorescent markers can be used to delineate tumor boundaries [16]. However, in contrast to glutamine uptake, cell membrane glutamate transport (GLAST1) is shifted from the cell membrane to an intracellular location in breast cancer cells as shown in **Figure 3**, effectively uncoupling extracellular glutamate from inhibiting the intracellular glutaminases; this allows full blown expression of intracellular glutamate generation(**Figure 1RXI**) and, if the relocated glutamate transporter, GLAST1 transports glutamate from the outer surface of inner mitochondrial membrane into the into the mitochondria matrix [17],

maintaining TCA pool homeostasis (anaplerosis).

transamination).

**Figure 1. GDH determines the fate of 15N glutamine**. Pathways of glutaminolysis, net keto acid production and NH4+ produced per glutamine consumed ratio. [1] Deamidation coupled to GDH deamination yielding 2NH4+/Gln and net keto acid (αKG). [2] Deamination coupled to ALT-mediated transamination yielding 1 NH4+/Gln and no net acid production. [3] Deamidation coupled to GDH reductive amination and transamination yielding < 1 NH4+/Gln and net keto acid
