**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],

Role of Glutamate Dehydrogenase in Cancer Growth and Homeostasis 33

then it would supply GDH glutamate in support of anaplerosis . Noteworthy overexpression of PIG promotes tumorigenesis [16] presumably by building up extracellular glutamate and suppressing local immune responses [18] . In addition NHE mediated acid extrusion is up-regulated in cancer cells [19,20] importing a Na+ load requiring Na+/K+ ATPase - ATP expenditure and ATP regeneration associated with acidogenic aerobic glycolysis(Warburg effect) and by substrate level phosphorylation. Because PIG (GGT/GGTP), NHE, glutamine transporter and glutaminase activities are all up-regulated in rapidly growing tumors, tagging molecular target inhibitors [21-24] with a ϒ-glutamyl moiety offers a tumor specific vehicle specific for limiting anaplerosis and preventing

**Figure 3. GLAST localization in normal versus cancer cells**. MCF10A and MCF7 cells cultured on coverglass were stained with monoclonal antibodies to GLAST-1 . MCF10A demonstrate almost complete membrane localization of the transporter, while MCF7 have a cytoplasmic distribution

Removal of glucose from the media (Figure 2, dotted gray line from GLC) deprives cells of pyruvate input into the TCA cycle and a fall in the intermediate (αKG) pool level[5] as reflected by a drop in glutamate [7]. As a consequence, GDH flux (**Figure 1, Rxn1**) increases [5] supplying anaplerosis as malate exits the cycle forming pyruvate which in turn supports citrate formation (**Figure 2**). Noteworthy this increased glutamate flux through GDH("pulling" effect) is maintained by 2 responses:1] by a small increase in glutaminase flux [5,7] and, 2] a large fall in glutamate transamination [5,7,25].Under glucose deprivation cell survival is dependent on GDH flux at least in part to supply anaplerosis [5,26]. Surprisingly cell number actually increase in the glutamine (1.3mM) alone compared to the glucose(5mM) plus glutamine media (**Figure 4A)** because of reduced cell death; this increased survival is attributed to the increased GDH flux [26]

**5. Glucose removal lowers TCA cycle intermediates and "pulls"** 

elevated cell pH, prerequisites for rapid tumor growth.

pattern.

**glutamate through GDH** 

**Figure 2. Central role of GDH flux in cancer cells**. Glucose **(GLC**) derived pyruvate**(PYR**) is metabolized(Warburg effect) to lactic acid(LAC) at the expense of the **TCA** (tricarboxylic acid) cycle intermediate pool(αKG) "pulling" glutamate through GDH to supply anaplerosis. **NHE(sodium hydrogen ion exchanger 1)** mediated acid extrusion is up-regulated coupled to anaerobic glycolysis acidifying extracellular milieu while cell membrane glutamate transporter(**GLAST1**) relocates to mitochondria with **PIG** produced GLU accumulating extracellularly and contributing with reduced pHe to host defense barrier. Glutamine(**GLN**) transported into cell by **ASC** and hydrolyzed to glutamate on outer surface of inner mitochondrial membrane by **GAC [13]** coupled with **GLAST1.** GLC removal (dashed line) accentuates GDH flux by "pull" mechanism while NHE mediated acid extrusion supported by GDH and accelerated αKG input with cytosolic **malate**(MAL) conversion to **PYR** supplying anaplerosis. TRO blocks PYR entry into mitochondria and accelerates GDH flux by exaggerated pull mechanism in conjunction with reduced pHi as result of NHE1 inhibition ("push" mechanism). **EGCG** inhibits GDH and induces cell death that can be partly rescued with methyl pyruvate(**CH3-PYR**) and restored TCA cycle pool while correction of cellular acidosis requires GDH flux pointing to the dual role for GDH in anaplerotic and acid base homeostasis.

then it would supply GDH glutamate in support of anaplerosis . Noteworthy overexpression of PIG promotes tumorigenesis [16] presumably by building up extracellular glutamate and suppressing local immune responses [18] . In addition NHE mediated acid extrusion is up-regulated in cancer cells [19,20] importing a Na+ load requiring Na+/K+ ATPase - ATP expenditure and ATP regeneration associated with acidogenic aerobic glycolysis(Warburg effect) and by substrate level phosphorylation. Because PIG (GGT/GGTP), NHE, glutamine transporter and glutaminase activities are all up-regulated in rapidly growing tumors, tagging molecular target inhibitors [21-24] with a ϒ-glutamyl moiety offers a tumor specific vehicle specific for limiting anaplerosis and preventing elevated cell pH, prerequisites for rapid tumor growth.

32 Dehydrogenases

**Figure 2. Central role of GDH flux in cancer cells**. Glucose **(GLC**) derived pyruvate**(PYR**) is metabolized(Warburg effect) to lactic acid(LAC) at the expense of the **TCA** (tricarboxylic acid) cycle intermediate pool(αKG) "pulling" glutamate through GDH to supply anaplerosis. **NHE(sodium hydrogen ion exchanger 1)** mediated acid extrusion is up-regulated coupled to anaerobic glycolysis acidifying extracellular milieu while cell membrane glutamate transporter(**GLAST1**) relocates to

pHe to host defense barrier. Glutamine(**GLN**) transported into cell by **ASC** and hydrolyzed to

supported by GDH and accelerated αKG input with cytosolic **malate**(MAL) conversion to **PYR** supplying anaplerosis. TRO blocks PYR entry into mitochondria and accelerates GDH flux by exaggerated pull mechanism in conjunction with reduced pHi as result of NHE1 inhibition ("push" mechanism). **EGCG** inhibits GDH and induces cell death that can be partly rescued with methyl pyruvate(**CH3-PYR**) and restored TCA cycle pool while correction of cellular acidosis requires GDH

flux pointing to the dual role for GDH in anaplerotic and acid base homeostasis.

glutamate on outer surface of inner mitochondrial membrane by **GAC [13]** coupled with **GLAST1.** GLC removal (dashed line) accentuates GDH flux by "pull" mechanism while NHE mediated acid extrusion

accumulating extracellularly and contributing with reduced

mitochondria with **PIG** produced GLU-

**Figure 3. GLAST localization in normal versus cancer cells**. MCF10A and MCF7 cells cultured on coverglass were stained with monoclonal antibodies to GLAST-1 . MCF10A demonstrate almost complete membrane localization of the transporter, while MCF7 have a cytoplasmic distribution pattern.
