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

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] which besides supporting anaplerosis also enhances NHE mediated acid extrusion (**Figure 4D**) although proliferation rate decreases (**Figure 4B**). Noteworthy is the increase in cell biomass(protein, nucleic acids and lipid) dependent upon glutaminolysis supported anaplerosis as shown by the increased incorporation of 14C-U-glutamine into cell biomass (**Figure 4C**).The critical role of GDH flux in cell survival is evident from the massive cell death induced by GDH inhibition under glucose depleted conditions with 100uM EGCG [5,26], an inhibitor of GDH [5]. Although supplying TCA cycle intermediates e.g. methyl pyruvate (10mM,**Figure 2**) rescued cells with GDH inhibited [5,26], a significant fraction of the population succumbed associated with a reduced cell pH [26]. Parenthetically, methylpyruvate is a strong acid constituting a large acid load which requires supplementing the media with equal moles of bicarbonate (10mM). Nevertheless, even after the above base compensation, supplying anaplerotic substrates does not restore NHE activity [26] pointing to an important dual role for GDH in maintaining both anaplerosis and pH homeostasis [22] for cell survival.

Role of Glutamate Dehydrogenase in Cancer Growth and Homeostasis 35

/glutamine)

**6. Cellular acidosis "pushes" glutamate through GDH** 

Glutamate flux through GDH can be also be "pushed" by a fall in intracellular pH [27]. Whether this reflects a shift from GHD1 to GDH2 isoform [28] is not known but, if so, this "pushing effect" of reduced pH effect could be additive with the above "pulling effect" of a reduced TCA pool (**Figure 2**). Indeed in metabolic acidosis, the ambient condition surrounding cancer cells in vivo, kidney cells' glutaminolysis is both "pushed"(reduced cell pHi, [27]) and "pulled"(inhibition of TCA, [29]) as a result of reduced TCA cycle pool size associated with true renal growth [30]. Interestingly enough, the in vivo kidney switches fuels from lactate to glutamine oxidation in metabolic acidosis[31] so that the anaplerotic glutaminolysis-GDH reactions matches [32] the cataplerotic reactions(CO2, biomass

generation. Furthermore the pH-dependent enlistment of GDH2 isoform alone (push mechanism) or accompanying GDH 1 flux (anaplerosis driven pull mechanism) would provide regulatory options in responding to anaplerotic/cataplerotic and, or, acid /base

Fortuitously there are agents that can be employed to impose this push/pull mechanism on the GDH flux in cancer cells and thereby present a window of vulnerability (targeted inhibitors). The antihyperglycemic agents, troglitazone (Rezulin) and rosiglitazone (Avandia) block pyruvate entrance into the TCA cycle(25,33] lowering αKG(glutamate,7,25] and accelerating GDH flux via this "pull" mechanism (**Figure 2**). Simultaneously, both troglitazone and rosiglitazone directly inhibit NHE [25,34] lowering pHi and driving GDH via the "push" mechanism (**Figure 2**). Noteworthy the glutaminase flux (glutamine disappearance) remains unchanged while the NH4+ production increases as the result of the increase in deamination flux (**GDH Figure 1, Rxn1**). Although resembling glucose deprivation ("pull" mechanism), troglitazone further increases the NH4+ production (additive "push+pull" mechanism) exceeding the fall in alanine production ("pull" mechanism alone). More specifically the accelerated GDH flux ("push+pull") induced by troglitazone can be demonstrated using 15N amino labeled glutamine as shown in **Figure 5**; in contrast, another glitazone pioglitazone(Actos) activates GDH flux [34] solely by reducing cell pH("push" mechanism) and consequently does not reduce alanine production [34]. Noteworthy troglitazone acutely inhibits GDH flux (0-3hrs) as the result of a fall in mitochondrial membrane potential( Ѱm) requiring accelerated GDH flux(3-24hrs) to fully restore the Ѱm [7], a response that is PPARγ independent [7,25] and possibly mitoNEET [35] dependent. Little recognized is the direct inhibition of NHE[20,34] by both troglitazone and rosiglitazone as well as indirect inhibition mediated through PPARγ suppression of NHE gene expression[20,36]; in contrast, pioglitazone does not inhibit NHE directly[34] rather acts indirectly through PPARγ[36]lowering cell pH[34] and accelerating GDH flux("push" mechanism). In combination, TRO +PIO together exert an additive effect on GDH flux

formation, [30,31] as does acid excretion (2NH4+/glutamine) and base(2HCO3-

**7. Glitazones accelerate GDH flux via the push/pull mechanism:** 

demands in tumors.

**A strategy for therapeutic intervention** 

**Figure 4.** Physiological(1.3mM) glutamine concentration alone supports breast cancer cell survival associated with increased anaplerotic and acid extrusion function. 4A GLN increased cell number and decreased cell death compared to GLN plus 5mM glucose; 4B Gln slows cell proliferation compared with GLC+GLN; 4C GLN supports anaplerotic function[14C-U-L-glutamine incorporation into TCA precipitated cellular protein and lipid 4D GLN supports accelerated NHE activity when assayed with K 10mM GLN as opposed to 10mM GLC
