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

34 Dehydrogenases

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

**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

anaplerosis and pH homeostasis [22] for cell survival.

10mM GLN as opposed to 10mM GLC

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